Peptide-enhanced transfections

ABSTRACT

The present invention provides compositions useful for transfecting eukaryotic cells comprising nucleic acid complexes with peptides, wherein the peptide is optionally covalently coupled to a nucleic acid-binding group, and cationic lipids or dendrimers as transfection agents. The invention also provides transfection compositions in which a peptide is covalently linked to the transfection agent (lipid, cationic lipid or dendrimer). Inclusion of peptides or modified-peptides in transfection compositions or covalent attachment of peptides to transfection agents results in enhanced transfection efficiency. Methods for the preparation of transfection compositions and methods of using these transfection compositions as intracellular delivery agents and extracellular targeting agents are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/911,569, filed Jul. 23, 2001, which is a continuation ofU.S. patent application Ser. No. 09/039,780, filed Mar. 16, 1998, nowPatent No. 6,376,248, which is a continuation-in-part of U.S. patentapplication Ser. No. 08/818,200, filed Mar. 14, 1997, now Patent No.6,051,429, which is a continuation-in-part of U.S. patent applicationSer. No. 08/658,130, filed Jun. 4, 1996, now U.S. Pat. No. 5,736,392,which is a continuation-in-part of U.S. patent application Ser. No.08/477,354, filed Jun. 7, 1995, now abandoned, all of which areincorporated by reference in their entirety herein to the extent notinconsistent with the disclosure herewith.

FIELD OF THE INVENTION

[0002] Compositions containing peptides, optionally conjugated tonucleic acid-binding groups, to lipids or to dendrimers, andtransfection agents, including cationic lipids and dendrimer polymers,useful for transfecting eukaryotic cells are disclosed. Also disclosedare methods of transfecting eukaryotic cells employing suchcompositions.

BACKGROUND OF THE INVENTION

[0003] Lipid aggregates such as liposomes can function to facilitateintroduction of macromolecules, such as DNA, RNA, and proteins, intoliving cells. Lipid aggregates comprising cationic lipid components canbe effective for delivery and introduction of large anionic molecules,such as nucleic acids, into certain types of cells. See Felgner, P.L.and Ringold, G.M. (1989) Nature 337:387-388 and Felgner, P. L. et al.(1987) Proc. Natl. Acad. Sci. USA 84:7413. Since the membranes of mostcells have a net negative charge, anionic molecules, particularly thoseof high molecular weight, are not readily taken up by cells. Cationiclipids aggregate to and bind polyanions, such as nucleic acids, tendingto neutralize the negative charge. The effectiveness of cationic lipidsin transfection of nucleic acids into cells is thought to result from anenhanced affinity of cationic lipid-nucleic acid aggregates for cells,as well as the function of the lipophilic components in membrane fusion.

[0004] Dendrimers are a new type of synthetic polymer with regular,dendric branching with radial symmetry composed of an initiator core,interior layers (or generations) of repeating units, radially attachedto the core and an exterior surface of terminal functional groups. See:D. A. Tomalia and H. D. Durst (1993) in E. Weber (ed.) Topics in CurrentChemistry, Vol. 165: Supramolecular Chemistry I-Directed Synthesis andMolecular Recognition, Springer-Verlag, Berlin, pp.193-313. The size,shape and surface charge density of the dendrimer is controlled bychoice of core, repeating unit, number of generations and terminalfunctional group. See: U.S. Pat. Nos. 5,527,524; 5,338,532; 4,694,064;4,568,737; 4,507,466; and PCT patent applications; WO8801179; WO8801178;WO9524221; and WO9502397. “STARBURST” (Trademark, Dendritech, Inc.) ordense star polyamidoamine dendrimers have been reported to mediateefficient transfection of DNA into mammalian cells (J. F.Kukowska-Latolla et al. (1996) Proc. Natl. Acad. Sci. USA 93:4897-4902and A. Bielinska et al. (1996) Nucleic Acids Res. 24(11):2176-2182).

[0005] “SUPERFECT” (Trademark, Qiagen, Inc.) or activated dendrimershave been reported to mediate efficient transfection of DNA intomammalian cells (J. Haensler and R. Szoka (1993) Bioconjugate Chem.4:372-379 and M. X. Tang et al., (1996) Bioconjugate Chem. 7P703-714).PCT patent application WO9524221 relates to bioactive or targeteddendrimer conjugates. PCT patent applications WO9319768 and WO9502397relate to polynucleotide delivery systems comprising dendrimers.

[0006] Transfection agents, including cationic lipids and dendrimers,are not universally effective for transfection of all cell types.Effectiveness of transfection of different cells depends on theparticular transfection agent composition and the type of lipidaggregate or dendrimer-complex formed. In general, polycationic lipidsare more efficient than monocationic lipids in transfecting eukaryoticcells. Behr, J -P. et al. (1989) Proc. Natl. Acad. Sci. 86:6982-6986,Hawley-Nelson, P., et al. (1993) FOCUS 15:73 and U.S. Pat. No. 5,334,761(Gebeyehu et al.). Behr et al. and EPO published application 304 111(1990), for example, describe improved transfection usingcarboxyspermine-containing cationic lipids including5-carboxyspermylglycine dioctadecyl-amide (DOGS) anddipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES). Thepolycationic liposomal transfection reagents 1,3dioleoyloxy-2-(6-carboxyspermyl)-propyl-amid (DOSPER,Boehringer-Mannheim) and “MULTIFECTOR” (Trademark, VennNova, Inc.) areother examples. For transfection, the optimal charge ratio ofDNA/dendrimer was found to be between 1:5 and 1:50 and G5 (generation5)-G10 (generation 10) dendrimers were reported capable of mediatingtransfection. Transfection efficiency of a given dendrimer varied withcell type, as has been observed with cationic lipid-mediatedtransfection (J. F. Kukowska-Latolla et al. (1996) Proc. Natl. Acad.Sci. USA 93:4897-4902).

[0007] Many biological materials are taken up by cells viareceptor-mediated endocytosis. See: Pastan and Willingham (1981) Science214:504-509. This mechanism involves binding of a ligand to acell-surface receptor, clustering of ligand-bound receptors, andformation of coated pits followed by internalization of the ligands intoendosomes. Both enveloped viruses, like influenza virus andalphaviruses, and non-enveloped viruses, like Adenovirus, infect cellsvia endocytotic mechanisms. See: Pastan, I. et al. (1986) in VirusAttachment and Entrv into Cells, (Crowell, R. L. and Lonberg-Holm, K.,eds.) Am. Soc. Microbiology, Washington, p. 141-146; Kielian, M. andHelenius, A. (1986) “Entry of Alphaviruses” in The Togaviridae andFlaviviridae, (Schlesinger, S. and Schlesinger, M. J., eds.) PlenumPress, New York p.91-119; FitzGerald, D. J. P. et al. (1983) Cell32:607-617. Enhancement of dendrimer-mediated transfection of some cellsby chloroquine (a lysosomotropic agent) suggests that endocytosis isinvolved in at least some dendrimer-mediated transfections.

[0008] Despite their relative effectiveness, however, successfultransfection of eukaryotic cell cultures using polycationic lipidreagents often requires high dosages of nucleic acid (approximately 10⁵DNA molecules per cell). The introduction of foreign DNA sequences intoeukaryotic cells mediated by viral infection is generally orders ofmagnitude more efficient than transfection with cationic lipid ordendrimer transfection agents. Viral infection of all the cells in aculture requires fewer than 10 virus particles per cell. Although thedetailed mechanism of fusion is not fully understood and varies amongviruses, viral fusion typically involves specific fusagenic agents, suchas viral proteins, viral spike glycoproteins and peptides of viral spikeglycoproteins. Vesicular stomatitis virus (VSV) fusion, for example, isthought to involve interaction between the VSV glycoprotein (G protein)and membrane lipids (Schlegel, R. et al. (1983) Cell 32:639-646). TheVSV G protein reportedly binds preferentially to saturable receptorssuch as acidic phospholipid phosphatidylserine (Schlegel, R. and M. Wade(1985) J. Virol. 53(1):319-323). Fusion of influenza virus involveshemagglutinin HA-2 N-terminal fusagenic peptides. See Kamata, H. et al.(1994) Nucl. Acids Res. 22(3):536-537.

[0009] Cell binding and internalization can also be enhanced,accelerated or made selective with peptides that bind cell receptors.For example, the penton-base protein of the Adenovirus coat contains thepeptide motif RGD (Arg—Gly—Asp) which mediates virus binding tointegrins and viral internalization via receptor-mediated endocytosis(Wickham, T. J. et al. (1995) Gene Therapy 2:750-756).

[0010] The efficiency of cationic lipid transfections has recently beenshown to be enhanced by the addition of whole virus particles to thetransfection mixture. See Yoshimura et al. (1993) J. Biol. Chem.268:2300. Certain viral components may also enhance the efficiency ofcationic lipid-mediated transfection. See: U.S. patent application Ser.No. 08/090,290, filed Jul. 12, 1993; and Ser. No. 08/274,397, filed Jul.12, 1994, now U.S. Pat. No. 5,578,475; incorporated by reference intheir entirety herein. The use of peptides from viral proteins toenhance lipid-mediated transfections was also recently suggested byKamata et al. (1994) Nucl. Acids Res. 22:536. Kamata et al. suggest that“LIPOFECTIN”-mediated transfections may be enhanced 3-4-fold by addinginfluenza virus hemagglutinin peptides to the transfection mixture.Despite these positive early indications, results vary as to theeffectiveness of including fusagenic or nuclear localization peptides inlipidic transfection compositions. Remy et al. (1995) Proc. Natl. Acad.Sci. USA 92:1744 report that “[a]ddition of lipids bearing a fusagenicor a nuclear localization peptide head group to the (polycationiclipid-DNA complex) particles does not significantly improve an alreadyefficient system.”

SUMMARY OF THE INVENTION

[0011] The present invention is based on the discovery that peptidesequences from viral, bacterial or animal proteins and other sources,including peptides, proteins or fragments or portions thereof cansignificantly enhance the efficiency of transfection of eukaryotic cellsmediated by transfection agents, including cationic lipids anddendrimers. The compositions and methods of the invention comprisepeptides, proteins and fragments thereof, modified peptides, modifiedproteins and modified fragments thereof, peptide conjugates, proteinconjugates and conjugates of fragments thereof, including those offusagenic, membrane-permeabilizing, receptor-ligand, and/ornuclear-localization peptides or proteins, or peptides or proteins thatlocalize to other sub-cellular locations (e.g., mitochondriallocalization peptides or proteins), which significantly improve theefficiency of transfection when bound to nucleic acid. In preferredembodiments, peptides, proteins, fragment thereof, or modified peptides,proteins and fragments thereof are bound or added to nucleic acid priorto adding the transfection reagent, although such peptides, proteins,fragments and modifications thereof may be added or complexed with thetransfection reagent prior to addition of the nucleic acid.Alternatively, the nucleic acid is combined with the transfection agentprior to addition of the peptide, protein, fragments and modificationsthereof. These fusagenic, receptor-ligand, nuclear localization,transport or trafficking, or other peptides can form a noncovalentassociation or complex with the nucleic acid that is to be introducedinto a cell. Complex formation can be enhanced by covalent coupling ofthe peptide or protein to a DNA-binding group, which can bind to thenucleic acid through conformational or charge interactions andfacilitate binding of the peptide to DNA. More generally, nucleicacid-peptide or protein complex formation can be enhanced by covalentcoupling of the peptide or protein to a nucleic acid-binding group.Nucleic acids (DNA and RNA and variants thereof) are more efficientlytransported into the cell by the transfection agent when bound topeptides or proteins of this invention and can with appropriate choiceof peptide or protein be directed to the cell nucleus or to othersub-cellular locations, thus requiring less nucleic acid startingmaterial.

[0012] This invention also relates to the covalent coupling of peptidesor proteins to the transfection agent, e.g., directly or via anappropriate linking or spacer group to a lipid of the cationic lipidtransfection composition (a cationic or neutral lipid) or directly orvia an appropriate linking or spacer group to a dendrimer. Of particularinterest are conjugated lipids and dendrimers that are covalently linkedto fusagenic peptides or proteins, transport or trafficking peptides orproteins, membrane-permeabilizing peptides or proteins andreceptor-ligand peptides or proteins. A variety of spacer groups may beused dependent upon the transfection agent and the peptide or protein.For example, spacers may be alkyl, ether, thioether, ester or amidegroups.

[0013] The cationic lipid compositions of the present invention and thedendrimer compositions of this invention provide significant advantagesover, prior art compositions, including enhanced transformationfrequency.

[0014] The present invention provides compositions and methods fortransfecting eukaryotic cells, particularly higher eukaryotic cells,with nucleic acids. Nucleic acids, both DNA and RNA, are introduced intocells such that they retain their biological function. Compositions fortransfecting eukaryotic cells comprising a peptide-nucleic acid complexor protein-nucleic acid complex and a transfection agent are provided.Transfection compositions of this invention include those in which thetransfection agent is any lipid, preferably a cationic lipid, a mixtureof cationic lipids or a mixture of cationic lipids and neutral lipids.Transfection compositions of this invention also include those in whichthe transfection agent is a dendrimer or mixture of dendrimers, as wellas mixtures of dendrimers and neutral or cationic lipids. Transfectioncompositions comprise a peptide or modified-peptide, e.g., apeptide-conjugate, or protein or fragment or portion thereof, modifiedor conjugated, which may bind nucleic acid and which are fusagenic,membrane-permeabilizing, or which function for nuclear localization,function for transport or trafficking, function for localization toanother sub-cellular location, and/or function as a receptor-ligand.Receptor-ligand peptides or proteins of this invention include thosethat bind to cell surface receptors, membrane receptors or cytosolicreceptors and that can function for cell targeting or cell adhesion, andinclude those that trigger internalization or endocytosis. The peptide-or protein-nucleic acid complex is formed by interacting a peptide orprotein or modified peptide or modified protein with nucleic acid or byinteracting the peptide or protein with a nucleic acid-transfectionagent complex. Modified peptides or proteins include peptides orproteins covalently conjugated to nucleic acid-binding groups. Peptide-or protein-conjugates of this invention also include peptide- orprotein-lipid (neutral or cationic) and peptide- or protein-dendrimerconjugates in which the peptide or protein is covalently linked to thetransfection agent or a component of the transfection agent.

[0015] For non-covalent peptide- or protein-enhanced lipid transfection,the peptide- or protein-nucleic acid complex is subsequently combinedwith a lipid, preferably a cationic lipid (or a mixture of a cationiclipid and neutral lipid) to form a peptide- or protein-nucleicacid-lipid aggregate which facilitates introduction of the anionicnucleic acid through cell membranes, including the nuclear membrane, ortargets the nucleic acid to a particular cell or sub-cellular location.Transfection compositions of this invention comprising peptide- orprotein-nucleic acid complexes and lipid can further include othernon-peptide agents that are known to further enhance transfection.

[0016] For lipid transfection employing a covalent peptide-orprotein-lipid conjugate, the peptide- or protein-lipid conjugate iscombined with nucleic acid, as is conventional for cationic lipidtransfection. The peptide- or protein-lipid conjugate may be firstcombined in a mixture of non-conjugated cationic and/or neutral lipidsand then combined with nucleic acid to form a peptide-orprotein-lipid-nucleic acid lipid aggregate which facilitatesintroduction of the anionic nucleic acid through cell membranes,including the nuclear membrane, or targets the nucleic acid to aparticular cell or to a sub-cellular location. Transfection compositionsof this invention comprising peptide- or protein-lipid conjugates andnucleic acids can further include other non-peptide or non-proteinagents that are known to further enhance transfection.

[0017] In an alternative transfection method of this invention employingfusagenic peptides or proteins covalently conjugated to lipids, thepeptide-or protein-lipid conjugate is complexed with non-conjugatedcationic lipids (or a mixture of cationic and neutral lipids). Asub-cellular localization peptide or protein, preferably a nuclearlocalization peptide or protein, is complexed to the nucleic acid andthe nucleic acid-peptide or protein complex is admixed with the cationiclipid-containing complex comprising covalently conjugated fusagenicpeptides or proteins. The resulting mixture exhibits enhancedtransfection efficiency.

[0018] For dendrimer transfection, the covalent peptide- orprotein-dendrimer conjugate is subsequently combined with nucleic acid,as is known in the art for dendrimer-mediated transfection, to form apeptide- or protein-dendrimer-nucleic acid aggregate that facilitatesintroduction of the anionic nucleic acid through cell membranes,including the nuclear membrane, or targets the nucleic acid to aparticular cell or sub-cellular location. When a peptide- orprotein-dendrimer conjugate is employed, the peptide or protein isbelieved, for the most part, to be concentrated at the outer surface ofthe dendrimer aggregate formed. Transfection compositions of thisinvention comprising peptide- or protein-dendrimer conjugates andnucleic acid can further include other non-peptide agents that are knownto further enhance dendrimer transfection, for example dendrimertransfection can be enhanced by addition of DEAE-dextran and/orchloroquin.

[0019] In alternative transfection compositions of this inventionemploying fusagenic peptides or proteins conjugated to dendrimers, thepeptide- or protein-dendrimer conjugate is admixed with a nucleic acidthat is itself complexed to a sub-cellular localization peptide orprotein, preferably a nuclear localization peptide or protein. The newcomplex (e.g., Sp-NLS-nucleic acid complexed to VSVG or RGD orE5-dendrimer) is optionally admixed with non-conjugated dendrimers oroptionally admixed with a cationic lipid-containing composition. Theresulting mixture exhibits enhanced transfection efficiency.

[0020] Peptides useful in transfection compositions include, but are notlimited to, functional portions of proteins and or polypeptides that arefusagenic, function for nuclear or other sub-cellular localization,function for transport or trafficking, are receptor ligands, comprisecell-adhesive signals, cell-targeting signals, cell-internalizationsignals or endocytosis signals as well as peptides or functionalportions thereof of viral fusagenic proteins, of viral nuclearlocalization signals, of receptor-ligands, of cell adhesion signals, ofcell-targeting signals or of internalization- or endocytosis- triggeringsignals. Peptides useful in this invention include naturally-occurringpeptides, peptides derived from synthetic or engineered proteins orpolypeptides, and synthetic analogs or functional equivalents ofnaturally-occurring peptides. Peptides of this invention include thosecomprised of the twenty commonly occurring amino acids, as well as rareamino acids, such as homocysteine and ornithine, or D-amino acids oramino acid analogs. Peptides and proteins or this invention can includepolyamines such as carboxy spermine. Transfection compositionscomprising viral peptides or functional portions of viral peptides ofinfluenza virus, vesicular stomatitis virus, adenovirus and simian virus40 are of particular interest. Transfecting compositions containingviral peptides (as well as proteins and polypeptides) modified so thatthey are covalently conjugated to DNA-binding groups, for example,spermine or related polyamines, are also useful in the methods of thisinvention.

[0021] Any proteins (or fragments or portions thereof) may be used inaccordance with this invention, either singly or in combination withother proteins or peptides. In a preferred aspect, two or more, three ormore, four or more, five or more, six or more, etc. proteins and/orpeptides are used in the invention. Additionally, such single ormultiple proteins and/or peptides may be used in combination with one ormore, two or more, three or more, four or more, five or more, six ormore, etc. transfection agents. In another preferred aspect, at leasttwo peptides and/or proteins are used in combination with a transfectionagent, preferably at least two transfection agents such as lipids and/ordendrimers.

[0022] Proteins useful in transfection compositions include, but are notlimited to, receptor ligands, membrane binding and fusion proteins,transport or trafficking proteins, nuclear localizing proteins, nuclearproteins, including proteins derived from chromatin, bacterialinternalization-mediating proteins, bacterial toxins or portions oftoxins (with toxin portion inactivated), which enter cells and localizeto subcellular compartments, membrane-disturbing proteins andantimicrobial proteins. Proteins include those derived from viral,bacterial, animal and other sources. Receptor-ligand proteins which areuseful include, but are not limited to, insulin, transferrin, epidermalgrowth factor, fibroblast growth factor, lactoferrin, and fibronectin.Useful viral membrane binding and fusion proteins include, but are notlimited to, the adenoviral proteins penton base, knob, and hexon, thevesicular stomatitis virus glycoprotein (VSVG), the coat proteins fromsemliki forest virus and the influenza hemagglutinin (HA). Viraltransport or trafficking proteins include, but are not limited to, HIVTat, hepatitis B virus core protein, and herpes simplex virus VP22. Alist of nuclear and chromatin proteins which are useful includes, but isnot limited to, the histone proteins, especially H1 and H2, the “highmobility group” proteins, especially HMG 1 and 17, protamine and hn RNPAl. Bacterial internalization proteins include, but are not limited to,invasin and internalin and proteins with similar functions derived fromListeria and Myobacterium tuberculosis. Bacterial toxins which entercells and localize to subcellular compartments include, but are notlimited to, Pseudomonas endotoxin A, Diphtheria toxin, and Shigellatoxin. In each case, the bacterial toxin function of these proteins andpolypeptides is inactivated to avoid detriment to transfected cells.Membrane-disturbing and anti-microbial proteins (some derived fromvenoms) include, but are not limited to, melittin, magainin, gramicidin,cecropin, defensins, protegrins, tachyplesins, thionins, indolicidin,bactenecin, drosomycin, apidaecins, cathelicidin,bacteriacidal/permeability-increasing protein (BPI), nisin, and buforin.

[0023] Inclusion of a peptide- or protein-nucleic acid complex or amodified peptide- or protein-nucleic acid complex in a cationic lipidtransfection composition can significantly enhance transfection (by2-fold or more) of the nucleic acid compared to transfection of thenucleic acid mediated by the cationic lipid alone. Enhancement ofdendrimer transfection by peptides or proteins or modified peptides ormodified proteins or fragments thereof is pronounced in a wide varietyof cell lines, including human primary cell lines and in cell lines thatare generally considered by those in the art to be “hard-to-transfect.”

[0024] Monovalent or polyvalent cationic lipids are employed in cationiclipid transfecting compositions. Preferred monovalent cationic lipidsare DOTMA (N-[1-(2.3-dioleoyloxy)-propyl]-N,N,N-trimethyl ammoniumchloride), DOTAP (1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane),DMRIE (1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammoniumbromide) or DDAB (dimethyl dioctadecyl ammonium bromide). Preferredpolyvalent cationic lipids are lipospermines, specifically DOSPA(2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoro-acetate) and DOSPER (1,3-dioleoyloxy-2-(6carboxyspermyl)-propyl-amid, and the di- and tetra-alkyl-tetra-methylspermines, including but not limited to TMTPS (tetramethyltetrapalmitoylspermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS(tetramethlytetralauryl spermine), TMTMS (tetramethyltetramyristylspermine) and TMDOS (tetramethyldioleyl spermine). Cationic lipids areoptionally combined with non-cationic lipids, particularly neutrallipids, for example lipids such as DOPE(dioleoylphosphatidylethanolamine), DPhPE(diphytanoylphosphatidylethanolamine) or cholesterol. A cationic lipidcomposition composed of a 3:1 (w/w) mixture of DOSPA and DOPE or a 1:1(w/w) mixture of DOTMA and DOPE are generally useful in transfectingcompositions of this invention. Preferred transfection compositions arethose which induce substantial transfection of a higher eukaryotic cellline.

[0025] Inclusion of a peptide- or protein-nucleic acid or modifiedpeptide- or protein-nucleic acid complex in a dendrimer transfectioncomposition can significantly enhance transfection (by 2-fold or more)of the nucleic acid compared to transfection of the nucleic acidmediated by the dendrimer alone or in combination with DEAE-dextran orchloroquine or both. Enhancement of transfection by peptides, proteins,modified peptides or modified proteins is pronounced in a wide varietyof cell lines, including human primary cell lines and in cell lines thatare generally considered by those in the art to be “hard-to-transfect.”

[0026] In general, any dendrimer that can be employed to introducenucleic acid into any cell, particularly into a eukaryotic cell, isuseful in the improved transfection compositions and methods of thisinvention. Dendrimers of generation 5 or higher (G5 or higher) arepreferred, with those of generation between G5-G10 being of particularinterest. Dendrimers of this invention include those with NH₃ orethylenediamine cores, GX(N1H₃) or GX(EDA), where X=the generationnumber. Dendrimers where X=5-10 being preferred. Dendrimers of thisinvention include those in which the repeating unit of the internallayers is a amidoamine (to form polyamidoamines, i.e. PAMAMs).Dendrimers of this invention include those in which the terminalfunctional groups at the outer surface of the dendrimer provides apositive charge density, e.g., as with terminal amine functional groups.The surface charge and the chemical nature of the outer dendrimersurface can be varied by changing the functional groups on the surface,for example, by reaction of some or all of the surface amine groups. Ofparticular interest are dendrimers that are functionalized by reactionwith cationic amino acids, such as lysine or arginine. Grafteddendrimers as described, for example in PCT applications WO 9622321 andWO9631549 and noted in U.S. Pat. No. 5,266,106, can be employed in thecompositions and methods of this invention. Activated dendrimers (J.Haensler and R. Szoka (1993) Bioconjugate Chem. 4:372-379 and M. X. Tanget al., (1996) Bioconjugate Chem. 7P703-714) can also be employed in thecomposition and methods of this invention.

[0027] The methods of the present invention involve contacting any cell,preferably a eukaryotic cell, with a transfection composition comprisinga peptide, a protein or fragment or portion thereof, including afusagenic, membrane-permeabilizing, transport or traffickingsub-cellular-localization, or receptor-ligand peptide or protein,optionally conjugated to a nucleic acid-binding group, or optionallyconjugated to the transfection agent (lipid or dendrimer) wherein saidpeptide or protein or modified peptide or protein is non-covalentlyassociated with the nucleic acid. In one embodiment, a peptide- orprotein-nucleic acid complex (where the peptide or protein can beconjugated to a nucleic-acid binding group) is formed and then combinedwith a cationic lipid for transfection. In a related embodiment, apeptide- or protein-lipid conjugate is combined optionally with otherlipids, including any appropriate cationic lipid, and then combined withnucleic acid for transfection. In another related embodiment, a nucleicacid-lipid complex is formed and then combined with a peptide or proteinfor transfection. In a second embodiment, a peptide- or protein-nucleicacid complex (where the peptide or protein can be conjugated to anucleic-acid binding group) is formed and then combined with a dendrimerfor transfection. In a related embodiment, a peptide-dendrimer conjugateis combined optionally with other dendrimers and then combined withnucleic acid for transfection. In another related embodiment, a nucleicacid-dendrimer complex is formed and then combined with a peptide orprotein for transfection. Dendrimers and/or peptide-conjugateddendrimers can be combined with cationic lipids and cationic lipidcomposition to obtain improved nucleic acid transfection compositions.In accordance with the invention, multiple peptides and/or proteins maybe added to accomplish transfection.

[0028] Methods of this invention employ among others, viral peptides orproteins of influenza virus, adenovirus, Semliki forest virus, HIV,hepatitis, herpes simplex virus, vesicular stomatitis virus or simianvirus 40 and more specifically an RGD-peptide sequence, an NLS peptidesequence and/or a VSVG-peptide sequence and to modified peptides orproteins of each of the foregoing. Methods of this invention areapplicable to transfection of adherent or suspension cell lines, ingeneral to animal cell lines, specifically to mammalian, avian,reptilian, amphibian and insect cell lines and more specifically toanimal primary cell lines, human primary cell lines, stem cell lines,and fibroblasts, as well as to cells in vivo in living organisms.

[0029] In one specific embodiment, a transfection-enhancing peptide orprotein is first bound to a nucleic acid to be introduced into a cell.The peptide- or protein-nucleic acid complexes are then admixed with atransfection agent (or mixture thereof) and the resulting mixture isemployed to transfect cells. Preferred transfection agents are cationiclipid compositions, particularly monovalent and polyvalent cationiclipid compositions, more particularly “LIPOFECTIN,” “LIPOFECTACE,”“LIPOFECTAMINE,” “CELLFECTIN,” DMRIE-C, DMRIE, DOTAP, DOSPA, and DOSPER,and dendrimer compositions, particularly G5-G10 dendrimers, includingdense star dendrimers, PAMAM dendrimers, grafted dendrimers, anddendrimers known as dendrigrafts and “SUPERFECT.”

[0030] In a second specific transfection method, atransfection-enhancing peptide or protein is conjugated to a nucleicacid-binding group, for example a polyamine and more particularly aspermine, to produce a modified peptide or protein which is then boundto the nucleic acid to be introduced into the cell. The modifiedpeptide-nucleic acid complex is then admixed with a transfection agent(or mixture thereof) and the resulting mixture is employed to transfectcells. In particular, the peptide or protein is covalently conjugated toa spermine, the spermine-modified peptide or protein is complexed withnucleic acid and admixed with a cationic lipid. Preferred transfectionagents are cationic lipid compositions, particularly monovalent andpolyvalent cationic lipid compositions, more particularly “LIPOFECTIN,”“LIPOFECTACE,” “LIPOFECTAMINE,” “CELLFECTIN,” DMRIE-C, DMRIE, DOTAP,DOSPA, and DOSPER, and dendrimer compositions, particularly G5-G10dendrimers, including dense star dendrimers, PAMAM dendrimers, grafteddendrimers, and including dendrimers known as dendrigrafts.

[0031] In a third specific embodiment, a mixture of one or moretransfection-enhancing peptides, proteins, or protein fragments,including fusagenic peptides or proteins, transport or traffickingpeptides or proteins, receptor-ligand peptides or proteins, or nuclearlocalization peptides or proteins and/or their modified analogs (e.g.,spermine modified peptides or proteins) or combinations thereof aremixed with and complexed with nucleic acid to be introduced into a cell.The peptide-nucleic acid complexes are then admixed with transfectionagent and the resulting mixture is employed to transfect cells.

[0032] In another specific embodiment, a component of a transfectionagent (lipids, cationic lipids or dendrimers) are covalently conjugatedto selected peptides, proteins, or protein fragments directly or via alinking or spacer group. Of particular interest in this embodiment arepeptides or proteins that are fusagenic, membrane-permeabilizing,transport or trafficking, or which function for cell-targeting. Thepeptide- or protein-transfection agent complex is combined with nucleicacid and employed for transfection.

[0033] The transfection compositions and methods of the presentinvention can be applied to in vitro and in vivo transfection of cells,particularly of eukaryotic cells, and more particularly to transfectionof higher eukaryotic cells, including animal cells. The methods of thisinvention can be used to generate transfected cells which express usefulgene products. The methods of this invention can also be employed as astep in the production of transgenic animals. The methods of thisinvention are useful as a step in any therapeutic method requiringintroduction of nucleic acids into cells including methods of genetherapy and viral inhibition and for introduction of antisense orantigene nucleic acids or ribozymes or RNA regulatory sequences orrelated inhibitory or regulatory nucleic acids into cells. Inparticular, these methods are useful in cancer treatment, in in vivo andex vivo gene therapy, and in diagnostic methods.

[0034] The transfection compositions and methods of this inventioncomprising peptides, proteins, peptide or protein fragments or modifiedpeptides or modified proteins, can also be employed as research reagentsin any transfection of eukaryotic cells done for research purposes. Thetransfection compositions can, with appropriate choice of physiologicmedium, be employed in therapeutic and diagnostic applications.

[0035] Transfection agents and transfection-enhancing agents of thisinvention can be provided in a variety of pharmaceutical compositionsand dosage forms for therapeutic applications. For example, injectableformulations, intranasal formulations and formulations for intravenousand/or intralesional administration containing these complexes can beused therapy.

[0036] In general the pharmaceutical compositions of this inventionshould contain sufficient transfection agent and any enhancing agents(peptide, protein, etc.) to provide for introduction of a sufficientlyhigh enough level of nucleic acid into the target cell or target tissuesuch that the nucleic acid has the desired therapeutic effect therein.The level of nucleic acid in the target cell or tissue that will betherapeutically effective will depend on the efficiency of inhibition orother biological function and on the number of sites the nucleic acidmust affect.

[0037] The dosage of transfection agent administered to a patient willdepend on a number of other factors including the method and site ofadministration, patient age, weight and condition. Those of ordinaryskill in the art can readily adjust dosages for a given type ofadministration, a given patient and for a given therapeutic application.

[0038] It will be appreciated by those of ordinary skill in the art thatthe transfection composition should contain minimal amounts ofinhibitory components, such as serum or high salt levels, which mayinhibit introduction of nucleic acid into the cell, or otherwiseinterfere with transfection or nucleic acid complexation. It will alsobe appreciated that any pharmaceutical or therapeutic compositions,dependent upon the particular application, should contain minimalamounts of components that might cause detrimental side-effects in apatient.

[0039] Components of the transfection compositions of this invention canbe provided in a reagent kit. In general, the kit comprises atransfection agent and a transfection-enhancing peptide, protein orfragment thereof. In one embodiment, a kit comprises individual portionsof cationic lipid and peptide, protein or fragment thereof or modifiedpeptide, protein or fragment thereof. In a second embodiment, a kitcomprises individual portions of dendrimer and peptide, protein orfragments thereof or modified peptide, protein or fragments thereof.Cationic lipid transfection kits can optionally include neutral lipid aswell as other transfection-enhancing agents or other additives, and therelative amounts of components in the kit may be adjusted to facilitatepreparation of transfection compositions. Kit components can includeappropriate medium or solvents for other kit components. Cationic lipidtransfection kits comprising a monocationic or polycationic lipidcomposition including a neutral lipid and a modified peptide or proteinare preferred. Dendrimer transfection kits can optionally include othertransfection enhancing agents, such as DEAE-dextran and/or chloroquine,as well as other additives and the relative amounts of components in thekit may be adjusted to facilitate preparation of transfectioncompositions. Dendrimer transfection kits comprising a G5-G10 dendrimeror a Lys- or Arg-modified dendrimer or dendrigraft or an activateddendrimer in combination with a peptide or protein or a modified peptideor protein are preferred. Kits provided by this invention include thosecomprising an individual portion of a polycationic lipid compositioncomprising DOSPA and DOPE or a monocationic lipid composition comprisingDOTMA and DOPE and a portion of modified peptide, particularly aspermine-modified peptide. Kits provided by this invention include thosecomprising an individual portion of a dendrimer and a portion of aspermine-modified peptide.

[0040] In related embodiments, kits of this invention can comprise apeptide- or protein-lipid conjugate or a peptide- or protein-dendrimerconjugate in combination with non-conjugated lipids, non-conjugateddendrimers and other agents to facilitate transfection.

[0041] Kits of this invention can include those useful in diagnosticmethods, e.g., diagnostic kits which in addition to transfection agentand transfection-enhancing agents (e.g., proteins, peptides, andfragments and modifications of peptides and proteins) can contain adiagnostic nucleic acid. A diagnostic nucleic acid is a general term forany nucleic acid which can be employed to detect the presence of anothersubstance (most generally an analyte) in a cell. For example, whentransfected into a cell a diagnostic nucleic acid may increase ordecrease expression of a gene therein in response to the presence ofanother substance in the cell (e.g., a protein, small molecule, steroid,hormone, or another nucleic acid). Diagnostic nucleic acids also includethose nucleic acids that carry some label or otherwise detectable markerto a particular target cell or target tissue for detection of the targetcell or tissue or for detection of a substance in the target cell ortissue.

[0042] Nucleic acids that can be transfected by the methods of thisinvention include DNA and RNA of any size from any source comprisingnatural bases or non-natural bases, and include those encoding andcapable of expressing therapeutic or otherwise useful proteins in cells,those which inhibit undesired expression of nucleic acids in cells,those which inhibit undesired enzymatic activity or activate desiredenzymes, those which catalyze reactions (ribozymes), and those whichfunction in diagnostic assays (e.g., diagnostic nucleic acids).Thereapeutic nucleic acids include those nucleic acids that encode orcan express therapeutically useful proteins, peptides or polypeptides incells, those which inhibit undesired expression of nucleic acids incells, those which inhibit undesired enzymatic activity or activatedesired enzymes in cells.

[0043] The compositions and methods provided herein can also be readilyadapted in view of the disclosure herein to introducebiologically-active macromolecules other than nucleic acids including,among others, polyamines, polyamine acids, polypeptides and proteinsinto eukaryotic cells. Other materials useful, for example astherapeutic agents, diagnostic materials, research reagents, which canbe bound to the peptides and modified peptides and introduced intoeukaryotic cells by the methods of this invention.

BRIEF DESCRIPTION OF THE FIGURES

[0044]FIG. 1 is a bar graph showing enhancement of transfection of humanfibroblast cells with various peptides added to “LIPOFECTAMINE”-DNAtransfection mixtures.

[0045]FIG. 2 is a graph showing the effect of Sp-NLSNLS concentration onenhancement of “LIPOFECTAMINE” transfection activity. Human fibroblastswere transfected with 0.4 μg DNA, 2 1L “LIPOFECTAMINE” and Sp-NLSNLS asindicated in serum-free medium. Cells were harvested and assayed forP-galactosidase activity 24 h after transfection.

[0046] FIGS. 3A-H provide comparisons of lipid transfection with andwithout Sp-NLSNLS precomplexed with DNA in 4 cell types. Humanfibroblast (3A and 3B); BHK-1(3C and 3D); NIH 3T3 (3E and 3F); MDCK (3Gand 3H) cells were transfected in 24-well plates with “LIPOFECTAMINE”and DNA as shown. FIG. 3A, C, E, and G: no Sp-NLSNLS; FIG. 3B, D, F andH: 1-4 μg Sp-NLSNLS/well, precomplexed with DNA.

[0047]FIGS. 4A and 4B are three-dimensional graphs comparing“LIPOFECTAMINE” transfection in the presence of varying amounts ofSp-NLSNLS with varying amounts of DNA (pSVneo) with transfection withoutpeptide. The transfections were performed in NIH 3T3 cells.

DETAILED DESCRIPTION OF THE INVENTION

[0048] The present invention provides improved methods for transfectingany cell, preferably eukaryotic cells with nucleic acids by employingpeptides, proteins, or fragments thereof, modified peptides or modifiedproteins, or modified fragments thereof, in combination withtransfection agents, e.g., cationic lipids and dendrimers. Theimprovement relates in one aspect to the use of a peptide- orprotein-nucleic acid complex to enhance the efficiency of cationiclipid-mediated or dendrimer-mediated transfection. The peptide- orprotein-nucleic acid complex comprises peptide bound to nucleic acid ora peptide modified to be covalently conjugated to a nucleic acid-bindinggroup which is then bound to nucleic acid. Alternatively, the peptide orprotein is used in combination with a nucleic acid-transfection agentcomplex. This invention has significant advantages over prior artmethods of transfection which employ cationic lipids or dendrimers fortransfection.

[0049] The peptides of this invention include fusagenic peptides,membrane-permeabilizing peptides, transport or trafficking peptides,nuclear localization peptides, and receptor-ligand peptides, amongothers. Receptor-ligand peptides include among others cell-adhesionpeptides, cell-targeting peptides, internalization-triggering peptides,and endocytosis-triggering peptides. Peptides useful in this inventioncan include peptide sequences functional for fusion (fusagenicsequences), transport , sub-cellular localization or which mediatebinding to a receptor. Peptides can include those that are functionalfragments of polypeptides or proteins, and may be synthetic or derivedfrom synthetic or engineered proteins or polypeptides. A peptide may bemulti-functional comprising sequences with more than one of thesefunctions. Peptides are optionally covalently coupled to anucleic-binding group, including a polyamine, and form a complex withthe nucleic acid. Peptide-complexed nucleic acids are more efficientlytransported into the cells and the cell nucleus, thus enhancing theefficiency of cationic lipid- or dendrimer-mediated cell transfection.

[0050] Because of the improved efficiency of transfection, considerablyless nucleic acid is required for effective transfection. Transfectioncompositions of this invention, by virtue of complex formation betweenthe nucleic acid and peptide or modified peptide, provide enhancedtransfection as compared to prior art cationic lipid and dendrimertransfection compositions.

[0051] The proteins of this invention include fusagenic proteins,membrane-permeabilizing proteins, transport or trafficking proteins,nuclear localization proteins, and receptor-ligand proteins, amongothers. Receptor-ligand proteins include among others cell-adhesionproteins, cell-targeting proteins, internalization-triggering proteins,and endocytosis-triggering proteins. Proteins useful in this inventioncan include peptide sequences functional for fusion (fusagenicsequences), transport, sub-cellular localization or which mediatebinding to a receptor. Proteins can include those that are functionalfragments of polypeptides or proteins, and may be synthetic orengineered proteins or comprise synthetic or engineered polypeptides. Aprotein may be multi-functional comprising sequences having more thanone of these functions. Proteins are optionally covalently coupled to anucleic-binding group, including a polyamine, and form a complex withthe nucleic acid. Protein-complexed nucleic acids are more efficientlytransported into the cells and the cell nucleus, thus enhancing theefficiency of cationic lipid- or dendrimer-mediated cell transfection.Because of the improved efficiency of transfection, considerably lessnucleic acid is required for effective transfection. Transfectioncompositions of this invention, by virtue of complex formation betweenthe nucleic acid and protein or modified protein, provide enhancedtransfection as compared to prior art cationic lipid and dendrimertransfection compositions.

[0052] Another aspect of this invention relates to improved efficiencyof transfection using peptide or protein conjugates in which a selectedpeptide (protein or protein fragment) is covalently linked to adendrimer or to a lipid that will be a component in a cationic lipidtransfection composition. The peptide- or protein-conjugatedtransfection agent is then employed in transfections as is known in theart for the non-conjugated transfection agent.

[0053] The following definitions are employed in the specification andclaims.

[0054] The term “transfection” is used herein generally to mean thedelivery and introduction of biologically functional nucleic acid into acell, e.g., a eukaryotic cell, in such a way that the nucleic acidretains its function within the cell. Transfection methods of thisinvention may be applied to cells in vitro or in vivo. The termtransfection includes the more specific meaning of delivery andintroduction of expressible nucleic acid into a cell such that the cellis rendered capable of expressing that nucleic acid. The term expressionmeans any manifestation of the functional presence of the nucleic acidwithin a cell, including both transient expression and stableexpression. Nucleic acids include both DNA and RNA without size limitsfrom any source comprising natural and non-natural bases. Nucleic acidscan have a variety of biological functions. They may encode proteins,comprise regulatory regions, function as inhibitors of gene or RNAexpression (e.g., antisense DNA or RNA), function as inhibitors ofproteins, function to inhibit cell growth or kill cells, catalyzereactions or function in a diagnostic or other analytical assay.

[0055] Transfection efficiency is “enhanced” when an improvement of atleast about 5 percent, preferably about 10 percent, and more preferablyabout 20 percent in efficiency is shown using the protocols formeasuring nucleic acid biological function set forth in the exampleshereof. Transfection is substantially enhanced when at least about a2-fold (i.e. 100% or more) improvement of efficiency is measured asdescribed herein.

[0056] The term “nucleic acid -binding group” is used herein generallyto mean a protein, peptide, polypeptide or polyamine which is capable ofnon-covalently associating with nucleic acids. Nucleic acid-bindinggroups include DNA-binding groups. Binding of the nucleic acid-bindinggroup to the nucleic acid can be specific to the sequence of the nucleicacid, or non-specific to its sequence. Although the mechanism ofassociation depends upon the particular binding group, sequencespecificity generally results from an ensemble of mutually favorableinteractions between a binding group and its target DNA. SomeDNA-binding groups, for example, interact with the DNA's paired basesand sugar-phosphate chains through direct contacts, including hydrogenbonds, salt bridges and van der Waals forces. Other groups functionthrough sequence-specific conformational variations in DNA (or moregenerally nucleic acid) rather than from sequence-specific hydrogenbonding interactions between nucleic acid and protein. It will beunderstood that the term “nucleic acid-binding group” includes anyprotein, peptide, polypeptide or polyamine which is capable of bindingnucleic acid, without regard to the mechanism of binding. Nucleicacid-binding groups are known to the art and widely available incommerce.

[0057] The term “peptide” as used herein is intended to be a genericterm which broadly includes short peptides (typically less than 100amino acids). Peptide used generically herein also includes peptidesmodified with nucleic acid-binding groups or peptides which retain aminoacid protecting groups, such as the Mtr group. Longer polypeptides(typically more than 100 amino acids), and proteins which contain one ormore polypeptide chains which function as transfection enhancing agentshaving fusagenic, cell-receptor ligand, transport or sub-cellularlocalization function, can be substituted for the peptides of thisinvention and can also be modified with nucleic acid-binding groups. Thepeptides of this invention typically have more than two amino acids;preferred peptides have more than 4 amino acids.

[0058] The peptides of this invention have biological function asfusagenic peptides, membrane-permeabilizing peptides,sub-cellular-localization peptides, cellular transport, andreceptor-ligand peptides. Two or more peptide functions can be combinedinto the same peptide, for example, by automated peptide synthesis.Peptides include dimers, multimers and contatimers of peptide sequencesthat have one or more desired functionalities.

[0059] The term spermine is used to describe the molecule spermine, butalso to describe peptides that are modified to be covalently linked to aspermine, as in the term “spermine-modified” peptide. Spermine may belinked directly or indirectly through intervening covalent bonds to thepeptide. Spermine-modified peptide can be used generically to describemodified peptides containing a linker to spermine. For example, the termspermine-modified also refers to peptides that are linked tocarboxyspermine.

[0060] Receptor-ligand proteins or peptides of this invention includethose peptides, proteins or protein fragments which bind to cell-surfaceor other membranes or which bind to soluble receptor molecules and whichoptionally have another biological function and which optionally triggerinternalization or endocytosis. Receptor-ligand proteins or peptidesinclude cell-adhesion proteins or peptides, and cell targeting proteinsor peptides.

[0061] Receptor-ligand proteins or peptides also include adhesionproteins or peptides. Adhesion proteins or peptides do not typicallytrigger endocytosis. Adhesion proteins or peptides include or can bederived from adhesion proteins including fibronectin, vitronectin,tenascin, laminins, collagens, thrombospondins, fibrinogens andfunctional equivalents. Such receptor-ligand peptides also includefragments of adhesion proteins including, but not limited to,fibronectin fragments such as “RETRONECTIN” (obtainable from Takara,Japan; see U.S. Pat. No. 5,198,423, which is incorporated by referencein its entirety herein). Table 1 provides examples of adhesion proteinsand peptides.

[0062] Fragments of adhesion proteins include RGD sequence-containingpeptides (RGD peptides) as listed in Table 1. The CS-1 peptide, sequencegiven in Table 1, is obtained from a 38 kD tryptic fragment of plasmafibronectin containing the carboxyl-terminal Heparin II domain and partof the type III connecting segment (IIICS) (Wayner, E. A. et al. (1989)“Identification and Characterization of T Lymphocyte Adhesion Receptorfor an Alternative Cell Attachment Domain (CS-1) in Plasma Fibronectin”J. Cell Biol. 109:1321-1330.)

[0063] Receptor ligand proteins or peptides also include those thattrigger internalization and/or endocytosis. For example, Penton Base isa pentamer coat protein of adenovirus that contains five copies of theintegrin receptor binding motif, Arg-Gly-Asp (RGD). Penton Base is usedby the virus to bind integrins α_(v)⊖₃ and α_(v)β₅. Following adenovirusattachment to cells by the fiber coat protein, the integrin receptorsmediate virus internalization in to the host cells. The Penton Base(wild-type) RGD sequence is HAIRGDTFAT [SEQ ID NO:1] (Wickham, T. J. etal. (1995) Gene Therapy 2:750-756.)

[0064] Adhesive peptides include RGD peptides which are peptidescontaining the tripeptide sequence Arg-Gly-Asp which can duplicate orinhibit the cell attachment promoting effects of fibronectin orvitronectin (Pierschbacher, M. D., and Ruoslahti, E. (1987) J. Biol.Chem. 262:17294-8), or other peptides with similar binding motifs.

[0065] Receptor-ligand proteins or peptides of this invention includethose proteins or peptides that have an affinity for or binding to,receptor molecules that are broadly expressed in a variety of celltypes, such as those proteins or peptides that bind to integrin α_(v)β₅.Receptor-ligand peptides of this invention also include those proteinsor peptides that bind to receptor molecules that are specificallyexpressed in a limited number of cell types (e.g. tissue-specific) orhighly expressed in a particular cell type (e.g., in cancer cells, suchas those that bind to the integrin α_(v)β₅, which is highly expressed incertain melanomas and glioblastoma).

[0066] Sub-cellular localization proteins or peptides include those thatrecognize, target or are directed to a particular sub-cellularcomponent, e.g., the nucleus, mitochondria, etc. See: C. Dingwall et al.(1991) TIBS 16:478-481.

[0067] Several proteins have been shown to be involved in transport ortrafficking within eukaryotic cells. This is an important cell functionfor the delivery of cellular components to their appropriatecompartments. Proteins that have the capability to cross cellularmembranes in reverse direction and reach the nucleus includeInterleukin-1 β, HIV Tat protein, acidic and basic fibroblast growthfactors, angiogenin, homeoprotein Antennapedia, Schwannoma derivedgrowth factor, and the Herpes Simplex Virus VP22 protein. These proteinsare able to cross the cell membrane and reach the nucleus. Two of theseproteins, HIV-Tat and HSV-VP22 have also been shown to mediate theuptake of other proteins when synthesized as fusions. The transportfunctions of the Tat protein were shown to be contained within an 11-12amino acid peptide, and fusions of heterologous proteins with thesepeptides were transported into cells. (Vives, E. et al. (1997), “Atruncated HIV-1 tat protein basic domain rapidly translocates throughthe plasma membrane and accumulates in the cell nucleus,” J. Biol. Chem.272:16010-16017; Kirsch, T. et al. (1996), “Cloning, high yieldexpression in Escherichia coli, and purification of biologically activeHIV-1 Tat protein,” Protein Expr. Purif. 8:75-84; Bonifaci, N. et al.(1995), “Nuclear translocation of an exogenous fusion protein containingHIV Tat requires unfolding,” AIDS 9:995-1000; Fawell, S. et al. (1994),“Tat-mediated delivery of heterologous proteins into cells,” Proc. Natl.Acad. Sci. (USA) 91:664-668; Pepinsky, R. B. et al. (1994), “Specificinhibition of a human papillomavirus E2 Trans-activator by intracellulardelivery of its receptor,” DNA and Cell Biol. 13:1011-1019; Mann, D. A.and Frankel, A. D. (1991), “Endocytosis and targeting of exogenous HIV-1Tat protein,” EMBO J. 10:1733-1739; Frankel, A.D.et al. (1989),“Activity of synthetic peptides from the Tat protein of humanimmunodeficiency virus type I,” Proc. Natl. Acad. Sci. (USA)86:7397-7401.

[0068] Most generally, any of the sequences exemplified in Tables 1-3,or functional equivalents thereof can be employed as peptides ormodified peptides to enhance transfection activity in the transfectioncompositions and methods of this invention. Specific examples ofspermine-modified peptides are provided in Table 4.

[0069] Table 2 lists a variety of peptides that are useful in thepresent invention, including peptides with a single functional regionand peptides combining two or more functional regions. Concatemers ofsingle function peptides and mixed concatemers combining sequentialrepetitions of dual (or more) function peptides are listed and areuseful in the present invention. The peptide formulas in Table 2 combinegeneric regions, e.g., spacers and linker groups with conserved aminoacid sequence associated with a particular function. Not specificallylisted in Table 2 are dimers and multimers of functional peptides, forexample, dimers formed between two cysteine residues of peptides. Dimersand multimers of functional peptides are useful in this invention.

[0070] Table 2 also lists cyclic peptides and cysteine peptideprecursors of cyclic peptides that contain a functional peptide sequenceof this invention (NLS, VSVG, RGD, LDV, E5, K5, etc.).

[0071] Table 3 provides a number of specific peptide sequences that areuseful in the methods and compositions of this invention for enhancementof transfection. The table includes a number of specific combinations oftwo functional peptide sequences with optional spacers and optionalcationic tails (for binding to nucleic acids). One entry in the table,“NLS phosphorylation” relates to an NLS sequence coupled to aphosphorylation-site-containing sequence. This fusion has been describedin H -P. Rihs et al. (1989) EMBO J. 8:1479-1484 and H—P Rihs et al.(1991) EMBO J. 10:633-639. The presence of the phosphorylation siteenhances transport to the nucleus.

[0072] Table 3 also contains precursors to transfection enhancingpeptides, HIS-TEV-peptides. These peptides contain a HIS tail and a TEV(Tobacco Etch Virus) protease recognition sequence in addition to thepeptide sequence useful for transfection enhancement. TEV protease willspecifically cleave the HIS tail from the peptide leaving the functionalpeptide sequence. This combination can be employed for the isolation andpurification of fusion polypeptides as has been described in U.S. Pat.No. 5,532,142 which is incorporated by reference herein. Peptides withHIS tails can be selectively purified on Ni columns.

[0073] Table 3 also includes examples of peptides having (D)_(n) tails,i.e., tails of anionic amino acids. These peptides are of particularinterest for binding to the surface of positively charged lipid-nucleicacid aggregates to enhance transfection of the aggregates and thenucleic acid that is carried therein. One transfection method for use ofthese peptides with anionic tails involves initial formation of cationiclipid aggregates with nucleic acid by conventional methods, followed bycomplexation to the anionic tailed peptide. A second transfection methodfor use of these peptides with anionic tails involves complexation ofanionic peptide tails to lipid, followed by addition of DNA. Thesepeptides can also be employed with dendrimer-nucleic acid complexes.

[0074] Those of ordinary skill in the art will appreciate that someamino acid sequence variation in functional peptides or modifiedpeptides, such as those listed in Tables 1, 2 and 3, can be toleratedwithout significant loss of function. In many cases, substitutions oflike amino acids, e.g., basic (cationic) amino acid for basic amino acid(e.g., K for R or R for K) or acidic (anionic) amino acid for acidicamino acid, in a given functional peptide will not significantly affectpeptide function. In functional peptides containing a string of likeamino acids, e.g., PKKKRKV [SEQ ID NO:2], addition or deletion of one ormore amino acids from the string may be tolerated, e.g., PKKKKRKV [SEQID NO:3], without significant loss of peptide function.

[0075] Generally, conservative amino acid substitutions or substitutionsof similar amino acids are tolerated without affecting protein orpeptide function. Similar amino acids can be those that are similar insize and/or charge properties, for example, Lysine and arginine,aspartate and glutamate and isoleucine and valine are pairs of similaramino acids. Similarity between amino acid pairs has been assessed inthe art in a number of ways. For example, Dayhoff et al. (1978) in Atlasof Protein Sequence and Structure, Volume 5, Supplement 3, Chapter 22,pages 345-352, which is incorporated by reference herein, providesfrequency tables for amino acid substitutions which can be employed as ameasure of amino acid similarity. Dayhoff et al.'s frequency tables arebased on comparisons of amino acid sequences for proteins having thesame function from a variety of evolutionarily different sources.Furthermore, for a given peptide, protein, or fragment thereof of thisinvention having a function for cell binding, adherence, cellinternalization, membrane permeabilization, nuclear localization andlike functions, there can be specific data currently and readilyavailable to the art regarding amino acid substitution that does noteffect peptide and/or protein function. All such peptides, proteins andfragments thereof having similar or conservative amino acidsubstitutions to the peptide, proteins and fragments thereof as listedin the Tables and description herein are encompassed by this inventionand re useful in the compositions and methods of this invention.Peptides, proteins and fragments thereof having similar or identicalfunction, for example for cell binding, cell adherence, cellinternalization, membrane permeabilization, nuclear localization andlike functions, to those listed in the Tables herein or described hereincan also be employed in the compositions and methods of this invention.

[0076] Variations that diverge the least from exemplified or art-knownfunctional peptide or protein sequences are generally preferred. For usein this invention, functional peptides can contain flanking strings ofamino acids (preferably glycines) that do not affect function of thecore peptide sequence. In an analogous way, a functional peptidesequence of this invention can be embedded within a larger peptide orprotein wherein the nature of the sequence external to the corefunctional sequence does not affect function of the core. This inventionincludes peptides which contain more than one distinct functionalsequence, e.g., NLSVSVG or RGDNLS. In these peptides, the functionalsequences can be separated by linker peptide regions (preferably one ormore Gs). Peptides of this invention can include amino acids that arenot part of a functional region which are added to the peptide toprovide a site for chemical linkage to another species, e.g., cysteinecan be used as a site for binding to spermine. In some cases, aminoacids external to the functional core sequence can act as spacers orlinker regions between the functional peptide and the species (lipid,dendrimer, polyamine, spermine, etc.) to which it is covalentlyattached. These amino acids may function in optimal configuration of thepeptide. For example, cysteine residues included in a peptide can beoxidized to form —S—S— dimers or larger multimer (trimers, etc.) byoxidization. Two cysteines placed distal to each other in a peptide canbe oxidized to prepare a cyclic peptide containing one or morefunctional amino acid sequences. A heterogenous dimer with greaterstability can be formed by incorporating penicillamine (Pen) in place ofcysteine (Pierschbacher et al. (1987) J. Biol. Chem. 262).

[0077] This invention also includes peptides or proteins containingfunctional groups that enhance transfection and also contain amino acidsor amino acid sequences that are useful in the preparation, isolationand purification of the peptides and proteins themselves. For example,aromatic amino acids can be included in a peptide or protein sequence toprovide a UV absorption marker to allow convenient measurement ofpeptide or protein concentration. Alternatively, transfection-enhancingpeptides and proteins can be provided with amino acid sequences thatspecifically bind to certain column materials to facilitate peptide orprotein purification. Certain transfection-enhancing peptides orproteins may be more easily produced through expression of DNA inbacteria or other expression systems. Peptides or proteins of thisinvention can include amino acid sequences (or parts thereof) that aresites for selective proteases that are useful in isolation of thepeptide or protein from an expression system.

[0078] The terms “modified-peptide” and “modified-protein” are usedherein generally to mean a peptide or protein which has been chemicallymodified to include a nucleic acid-binding group covalently attachedthereto. The term “modified-peptide” as used herein includes“polyamine-peptide conjugate” wherein the covalently attached nucleicacid-binding, or more specifically a DNA-binding group, is a polyamine,including “spermine-modified peptide” wherein the DNA-binding group isspermine. In some cases, a peptide or protein may itself bind to nucleicacid; in other cases modification of the peptide is necessary for orenhances binding to nucleic acid. For example, strings of cationic aminoacids can be added to a functional peptide (at the C- or N-terminus) tofacilitate binding to nucleic acid. These sequences can be written as(Uaa)_(u), where u is an integer ranging typically from 1 to about 20(and more preferably ranges from 8-20) and Uaa, independently of otherUaa's in the peptide, is a cationic amino acid, e.g., (K)_(u)[SEQ IDNO:4], (R)u [SEQ ID NO:5], or (KR)_(u)[SEQ ID NO:6]. More generally,cationic amino acid strings for binding to nucleic acids can includenon-cationic amino acids (preferably Gs) so long as the binding functionis not significantly decreased.

[0079] Naturally-occurring peptides or proteins may require additionalmodification to allow conjugation to spermine or other polyamines. Forexample, cysteines may be added to the C-terminals or N-terminals ofpeptides or introduced within a peptide to facilitate conjugation.Likewise a string of spacer amino acids, i.e. (G)_(n) [SEQ ID NO:7],where n is an integer ranging most generally from 1-about 20, can beadded between a peptide and the species to which it is covalentlylinked. Any peptide modification used to facilitate conjugationpreferably does not substantially affect peptide binding or function.

[0080] The term “peptide-nucleic acid complex” generally refers to thenoncovalent association between a peptide or protein and a nucleic acid.The peptide or protein of this complex may be a modified peptide asdefined above. As used herein in certain embodiments of transfectionmethods, a “peptide-nucleic acid complex” is formed prior to theaddition of cationic lipid or dendrimer to a transfection composition.

[0081] “Lipid aggregate” is a generic term that includes liposomes ofall types both unilamellar and multilamellar as well as vesicles,micelles and more amorphous aggregates. A cationic lipid aggregate is alipid aggregate comprising sufficient cationic lipid, optionally incombination with non-cationic (e.g., neutral) lipids, such that thelipid aggregate has a net positive charge. Cationic lipids and lipidaggregates are capable of aggregating the peptide-nucleic acid complexesof the invention.

[0082] Cationic lipid composition includes those compositions comprisinga cationic lipid or a mixture of cationic lipids, which can be eithermonovalent or polyvalent cationic lipids. The cationic lipid compositionoptionally contains neutral lipids. Of particular interest are cationiclipid compositions recognized in the art as useful in transfectionmethods. Preferred cationic lipid compositions comprise monovalent orpolyvalent cationic lipids; more preferred are those compositionscontaining DOTMA, DOTAP, DDAB, DMRIE, DOSPA, DOSPER, TMTPS and theiranalogs or homologs; the most preferred cationic compositions are“LIPOFECTIN” and “LIPOFECTAMINE.”

[0083] Transfection activity or efficiency is measured by detecting thepresence of the transfected nucleic acid in a cell. This is oftenassessed by measuring the biological function of the nucleic acid in thecell, and most often assessed by measuring the level of transient orstable expression of a reporter gene comprised in the transfectednucleic acid. Reporter gene expression depends among other things on theamount of nucleic acid transfected as well as promoter function in thecell. Transfection activity can also be assessed by determining thepercent of cells in a sample that have been transfected, for example, byassessing reporter gene expression using cell counting or in situstaining methods. The transfection methods of this invention employingpeptides in combination with cationic lipids can display significantenhancement of transfection (2-fold or more) over transfection methodsemploying comparable cationic lipids alone.

[0084] The method of this invention involves contacting a eukaryoticcell with a transfection composition comprising a peptide-nucleic acidcomplex (or a modified peptide-nucleic acid complex) and a transfectionagent, a cationic lipid or a dendrimer. A cationic lipid transfectioncomposition optionally comprises a non-cationic lipid, preferably aneutral lipid. Cationic lipid transfection compositions can optionallycomprise known transfection enhancing agents in addition to peptides ormodified peptides, including, for example chloroquine, a lysosomotrophicagent. Dendrimers or mixtures thereof can be employed in transfectioncompositions. Dendrimer transfection compositions may include agentsother than peptides or modified peptides that are known to enhancedendrimer-mediated transfection, e.g., DEAE-dextran and/or chloroquine.The peptide or protein can be a fusagenic peptide or protein of a virus.A preferred fusagenic peptide or protein is that of influenza virushemagglutinin or vesicular stomatitis virus G-protein, or VSVG. Thepeptide or protein can be a sub-cellular localization signal peptide orprotein. A preferred nuclear localization signal peptide is that ofsimian virus 40, particularly the nuclear localization sequence (NLS) ofthe SV40 large T antigen (Kalderon et al. (1984) Cell 39:499; andLanford et al. (1986) Cell 46:575). There is some diversity in thesequences of nuclear localization signals as reported in C. Dingwell andR.A. Laskey (1991) TIBS:478-481, which is incorporated by referenceherein for the sequences disclosed. This invention includes peptides orproteins comprising nuclear localization sequences as disclosed therein.The peptide or protein of this invention can be a receptor-ligandpeptide or protein. Preferred receptor-ligand peptides are cell adhesionpeptides, particularly RGD peptides or other integrin-binding peptides.Transfecting compositions comprising peptides or proteins of viralproteins conjugated to a nucleic acid-binding group are particularlypreferred. Preferred nucleic acid-binding groups are spermines and thecationic amino acid strings (K)_(u) and (R)_(U) where u is an integerfrom 1 to about 20 and more preferably is about 8 to about 20.

[0085] Enhanced transfection methods of this invention are demonstratedwith the prototype nuclear localization signal peptide from simian virus40 and the prototype fusagenic peptides from influenza (HApep; E5 and K5amphophilic peptides), vesicular stomatitis virus (G protein) and an RGDpeptide (GRGDSPC, SEQ ID NO:8) taken from the cell attachment site offibronectin. The DNA-binding group that has been employed is a polyaminecapable of forming a noncovalent association with the base pairs of thenucleic acid. Enhanced transfection methods of this invention have beenfurther exemplified using the prototype DNA-binding group, spermine.

[0086] In some cases, the peptides or proteins form a direct noncovalentassociation or complex with the nucleic acid. This peptide-nucleic acidcomplex forms as a consequence of conformational or charge interactionsbetween the peptide and the base pairs of the DNA. A peptide-nucleicacid complex forms spontaneously in an appropriate medium. Transfectioncompositions comprising these peptide-nucleic acid complexes areprepared by first interacting the nucleic acid with the peptide orprotein followed by addition of the resulting complex to a cationiclipid composition or a dendrimer.

[0087] The peptides or proteins of this invention, when covalentlycoupled to a nucleic acid-binding group (modified-peptide), can form anoncovalent association or complex with the nucleic acid. Thismodified-peptide-nucleic acid complex forms as a consequence ofconformational or charge interactions between the nucleic acid-bindinggroup and the nucleic acid (DNA or RNA). For example, the prototypespermine-peptide-nucleic acid complex likely forms as a consequence ofcharge interactions between the amines of spermine and the phosphates onthe DNA backbone. A modified-peptide-nucleic acid complex formsspontaneously in an appropriate medium. Transfection compositionscomprising these modified-peptide-nucleic acid complexes are prepared byfirst interacting the nucleic acid with the modified peptide to formcomplexes followed by addition of a cationic lipid composition.

[0088] In one embodiment, a composition containing the peptide-nucleicacid or modified-peptide-nucleic acid complex is admixed with a cationiclipid, alone or in combination with a non-cationic lipid, to form apeptide-nucleic acid-lipid aggregate. A peptide-nucleic acid-lipidaggregate forms spontaneously in an appropriate medium or variouswell-known techniques may also be employed to produce a desired type oflipid aggregate. The relative amounts of cationic lipid and non-cationiclipid employed depends on a number of factors, including the cell typeto be transfected, toxicity of the lipids to the cell and theenvironment (e.g., medium) in which the aggregate is to be employed. Thekinds and amounts of lipids employed is typically balanced for a givencell type to minimize cell toxicity and maximize transfectionefficiency.

[0089] In another embodiment, peptide-nucleic acid ormodified-peptide-nucleic acid complexes are admixed with a dendrimer (ormixture of dendrimers) to form a peptide-nucleic acid-dendrimeraggregate. This aggregate forms spontaneously in an appropriate medium.The relative amounts of dendrimer to nucleic acid are adjusted tooptimize transfection in a given cell type in a given environment. Thechemical type, size and shape of the dendrimer is also selected tooptimize transfection in a given cell type.

[0090] Nucleic acid delivery can be enhanced by the use of celltargeting, cell adhesion or binding peptides or proteins. Peptidescontaining the RGD sequence can be coupled to the polycation sperminewhich acts as a DNA binding group. The RGD-spermine peptide is believedto enhance transfection via cell targeting, and more importantly, celladhesion. Attachment to adhesion proteins, and in some cases to othercells, is often mediated by integrins. Many adhesive proteins present inextracellular matrices and in the blood contain the tripeptidearginine-glycine-aspartic acid (RGD), as their cell recognition site(Ruoslahti, E. and Pierschbacher, D. (1987) Science 238:491). Pathogenssuch as bacteria, and more specifically, foot and mouth disease virus(FMDV) (Mason et al. (1994), Proc. Natl. Acad. Sci. 91, 1932-1936) andAdenovirus (Wickham, T. J. et al (1995), “Targeting of adenovirus pentonbase to new receptors through replacement of its RGD motif with otherreceptor-specific peptide motifs,” in Gene Therapy 2-750-756) have RGDcontaining proteins expressed on their surface, which interact withintegrins on the host cell and facilitate internalization. RGD, (K)u RGD[SEQ ID NO:9](particularly where u=16 [SEQ ID NO: 10]) and RGD-sperminepeptide can enhance “LIPOFECTIN-,” “LIPOFECTAMINE-” or DOSPER-mediatedtransfection or dendrimer-mediated transfections.

[0091] Viral peptides or proteins can be isolated by a variety ofwell-known techniques, for example using the cationic detergent DTAB asdescribed in Glushakova, S. E., et al. (1985) “Influenza viralglycoproteins isolation using cationic detergent dodecylmethylammoniumbromide and its subsequent internalization into liposomal membrane” Mol.Genet. Microbiol. Virol. 4:39-44. Alternatively, viral peptides orproteins, as well as functional peptides or proteins from other sources,can be produced by a variety of standard chemical synthetic methods.Functional peptides, for example, can be synthesized using automatedsolid phase peptide synthesis as described, e.g., in Stewart et al.(1984) Solid Phase Peptide Synthesis, Pierce Chemical Company, Rockford,Ill. Fusagenic peptides from influenza and vesicular stomatitis virus,including the exemplified hemagglutinin peptide, K5 and E5 amphophilicpeptides and G protein, are particularly useful in the methods of thisinvention. Nuclear localization signal peptides from simian virus 40,including the exemplified NLS peptide, are also preferred. Peptides orproteins can be used alone or in combination with other functionalpeptides or proteins in the methods of this invention. As illustrated inTable 2, two or more functional peptide sequences (optionally separatedby linkers or spacers) can be combined in a given peptide.

[0092] Modified-peptides or proteins can be prepared by a variety ofwell-known coupling techniques, for example using a heterobifunctionalcross-linking agent as described in the Examples hereof. A variety ofcross-linking agents are known to the art and widely available incommerce including, without limitation, succinimidyl or maleimidylcross-linkers, such as Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate(Sulfo-SMPB), disuccinimidyl suberate, succinimidyl4-(p-maleimidophenyl)butyrate (SMPB),4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)-toluene (SMPT),Sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate(Sulfo-LC-SPDP), Succinimidyl6-[3-(2-pyridyldithio)propionamido]hexanoate (LC-SPDP), N-Succinimidyl3-(2-pyridyldithio)propionate (SPDP), Sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC),Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC);m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester (Sulfo-MBS),m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS),Sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (Sulfo-SIAB),N-Succinimidyl(4-iodoacetyl)aminobenzoate (SIA). Methods for conjugatingpeptides or proteins and polyamines are well-known in the art.Representative methods are disclosed in Staros, J. V. (1982)Biochemistry 21:3990. Any of the functional peptides or proteinsexemplified herein, or functional equivalents thereof, can be modifiedby covalent coupling to a nucleic acid-binding agent, e.g., to apolyamine and preferably to spermine.

[0093] Covalent linking of a peptide or protein to a lipid or adendrimer can be performed by a variety of conventional methods usingknown coupling agents and known derivatization methods.

[0094] Media employed in transfections should preferably be free ofcomponents, like serum or high salt levels, that can inhibit cationiclipid-mediated or dendrimer-mediated transfection of cells.

[0095] A variety of cationic lipids is known in the art. Generally, anycationic lipid, either monovalent or polyvalent, can be used in thecompositions and methods of this invention. Polyvalent cationic lipidsare generally preferred. Cationic lipids include saturated andunsaturated alkyl and alicyclic ethers and esters of amines, amides orderivatives thereof. Straight-chain and branched alkyl and alkene groupsof cationic lipids can contain from 1 to about 25 carbon atoms.Preferred straight-chain or branched alkyl or alkene groups have six ormore carbon atoms. Alicyclic groups can contain from about 6 to 30carbon atoms. Preferred alicyclic groups include cholesterol and othersteroid groups. Cationic lipids can be prepared with a variety ofcounter ions (anions) including among others: Cl⁻, Br⁻, I⁻, F⁻, acetate,trifluoroacetate, sulfate, nitrite, triflate, and nitrate.

[0096] A well-known cationic lipid isN-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA).See Felgner, P. L. et al. (1987) Proc. Natl. Acad. Sci. USA84:7413-7417. DOTMA and the analogous diester DOTAP(1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane) are commerciallyavailable. Additional cationic lipids structurally related to DOTMA aredescribed in U.S. Pat. No. 4,897,355, which is incorporated by referencein its entirety herein.

[0097] Other useful groups of cationic lipids related to DOTMA and DOTAPare commonly called DORI-ethers or DORI-esters. DORI lipids differ fromDOTMA and DOTAP in that one of the methyl groups of thetrimethylammonium group is replaced with a hydroxyethyl group. The DORIlipids are similar to the Rosenthal Inhibitor (RI) of phospholipase A(Rosenthal, A. F. and Geyer, R. P. (1960) J. Biol. Chem. 235:2202-2206).The oleoyl groups of DORI lipids can be replaced with other alkyl oralkene groups, such as palmitoyl or stearoyl groups. The hydroxyl groupof the DORI-type lipids can be used as a site for furtherfunctionalization, for example for esterification and/or for etherformation.

[0098] Additional cationic lipids which can be employed in thecompositions and methods of this invention include those described asuseful for transfection of cells in PCT application WO 91/15501published Oct. 17, 1991, Pinnaduwage, P. et al. (1989) Biochem. Biophys.Acta. 985:33-37; Rose, J. K. et al. (1991) BioTechniques 10:520-525;Ito, A et al. (1990) Biochem, Intern, 22:235-241.

[0099] The polycationic lipid formed by conjugating polylysine to DOPE(Zhou, X. et al. (1991) Biochem. Biophys. Acta 1065:8-14), as well asother lipopolylysines, can also be employed in the methods andcompositions of this invention.

[0100] Polycationic lipids containing carboxyspermine are also useful inthe compositions and methods of this invention. Behr, J-P. et al. (1989)Proc. Natl. Acad. Sci. 86:6982-6986 and EPO published application 304111 (1990) describe carboxyspermine-containing cationic lipids including5-carboxyspermylglycine dioctadecyl-amide (DOGS) anddipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES).Additional cationic lipids can be obtained by replacing the octadecyland palmitoyl groups of DOGS and DPPES, respectively, with other alkylor alkene groups. Polycationic lipids designated DOSPER (See: Formula Bfor specific and generic formula) are also useful in the methods of thisinvention. U.S. Pat. No. 5,334,761, which is incorporated by referencein its entirety herein, also describes cationic lipids, including DOSPA(see: Formula A for specific and generic formula) which are useful inthis invention. Also, U.S. Pat. No. 5,674,908, which is incorporated byreference in its entirety herein, describes polycationic lipids,including TMTPS, which are useful in this invention.

[0101] PCT application WO 95/17373 describes highly packed polycationicammonium, sulfonium and phosphonium lipids that are useful fortransfection. These cationic lipids are useful in methods of thisinvention.

[0102] In the transfection compositions of this invention cationiclipids can optionally be combined with non-cationic lipids, preferablyneutral lipids, to form lipid aggregates that bind to themodified-peptide-nucleic acid complex. Neutral lipids useful in thisinvention include, among many others: lecithins;phosphotidylethanolamine; phosphatidylethanolamines, such as DOPE(dioleoylphosphatidylethanolamine), DPhPE(diphytanoylphosphatidylethanolamine), DPPE(dipalmitoylphosphatidylethanolamine),dipalmiteoylphosphatidylethanolamine, POPE(palmitoyloleoylphosphatidylethanolamine) anddistearoylphosphatidylethanolamine; phosphotidylcholine;phosphatidylcholines, such as DOPC (dioleoylphosphidylcholine), DPPC(dipalmitoylphosphatidylcholine) POPC(palmitoyloleoylphosphatidylcholine) and distearoylphosphatidylcholine;phosphatidylglycerol; phosphatidylglycerols, such as DOPG(dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidyl-glycerol),and distearoylphosphatidylglycerol; phosphatidylserine;phosphatidylserines, such as dioleoyl- or dipalmitoylphosphatidylserine;diphosphatidylglycerols; fatty acid esters; glycerol esters;sphingolipids; cardolipin; cerebrosides; and ceramides; and mixturesthereof. Neutral lipids also include cholesterol and other 3βOH-sterols.

[0103] Dendrimers can be prepared by several now well-documentedmethods. See: WO95/24221; D.A. Tomalia and H.D. Durst (1993) in E. Weber(ed.) Topics in Current Chemistry, Vol. 165: Supramolecular ChemistryI-Directed Synthesis and Molecular Recognition, Springer-Verlag, Berlin,pp.193-313; U.S. Pat. Nos. 5,527,524; 5,338,532; 4,694,064; 4,568,737;4,507,466; and PCT patent applications; WO8801179; WO8801178; andWO9502397. “STARBURST” (Trademark, Dendritech, Inc.) or dense starpolyamidoamine (PAMAM) dendrimers including those having cationic aminoacids or other cationic species at their outer surface or “SUPERFECT”(Trademark, Qiagen, Inc.) or activated dendrimers are preferred fortransfection methods of this invention. Transfection protocols for usewith dendrimers and a discussion of the choice of a given dendrimer fora given transfection is given in J. F. Kukowska-Latolla et al. (1996)Proc. Natl. Acad. Sci. USA 93:4897-4902; A. Bielinska et al. (1996)Nucleic Acids Res. 24(11):2176-2182; WO9524221; WO9319768; WO9502397;and J. Haensler and R. Szoka (1993) Bioconjugate Chem. 4:372-379 and M.X. Tang et al., (1996) Bioconjugate Chem. 7P703-714.

[0104] The present invention is based on the discovery that certainpeptides or proteins or modified peptides or proteins can significantlyenhance the efficiency of transfection of eukaryotic cells with nucleicacids. The peptide or protein or modified peptide or protein binds tothe DNA and functions as a fusagenic peptide or protein, functions forsub-cellular localization or for cell adhesion. Peptides or proteins,optionally modified, if necessary or desirable, to enhance binding tonucleic acids, that function as internalization-triggering signals orendocytosis-triggering signals or transport signals, also function inthe transfection methods of this invention. The compositions and methodsof the invention comprise peptides or proteins, optionally modifiedcovalently with a nucleic acid-binding group, which significantlyimprove the efficiency of transfection when bound to nucleic acid priorto adding the transfection reagent. These bound nucleic acids are moreefficiently transported into the cell and to the cell nucleus, thusrequiring less nucleic acid starting material. Although the presentinvention is exemplified using a cationic lipid delivery system or adendrimer delivery system, fusagenic, sub-cellular localization peptidesand cell-targeting peptides are effective in enhancing transfectionusing a variety of known delivery systems. The present invention thusprovides improved methods of transfection using these peptides andmodified peptide, including peptides covalently conjugated to dendrimersor peptides covalently conjugated to lipids, to enhance transfection byother nucleic acid delivery means including, without limitation,electroporation (T. K. Wong and E. Neumann (1982) Biochem. Biophys. Res.Commun. 107:584 and E. Neumann et al. (1982) EMBO J. 1:841), calciumphosphate (F. L. Graham and A. J. Vander Eb (1973) Virology 52:456),microinjection (M. R. Capecchi (1920) 22:479), ballistic transformationusing microscopic particles coated with DNA (D. T. Tomes et al. (1990)Plant Mol. Biol. Manual A13:1-22 and G. N. Ye et al. (1990) Plant.Molec. Biol. 15:809) DEAE-dextran (A. Vaheri and J. S. Pagano (1965)Science 175:434), and polybrene-DMSO(S. Kawai and M. Nishizawa (1984)Molec. Cell. Biol. 4:1172).

[0105] Transfection compositions of this invention include compositionsfor transfecting eukaryotic cells using a peptide or protein comprisinga nuclear localization sequence, a fusagenic peptide or transportpeptide, receptor-ligand peptide or transport peptide sequencecovalently attached to a polycation. Peptides or proteins having anuclear localization sequence, fusagenic peptide, transport peptide orreceptor-ligand signal attached to a polycation, are also a part of theinvention. Preferred linkers include, for example, heterobifunctionalcrosslinkers. The polycation is preferably a polyamine and mostpreferably, spermine. As previously discussed, the transfectioncompositions and peptides of the invention are useful with a widevariety of delivery systems including, without limitation,electroporation, calcium phosphate, microinjection, ballistictransformation, DEAE-dextran and polybrene-DMSO. The present inventionthus includes methods for transfecting a eukaryotic cell with a nucleicacid, the method generally comprising the steps of (1) admixing apeptide or protein or modified peptide or protein with a nucleic acid toform a peptide-nucleic acid complex; and (2) introducing thepeptide-nucleic acid complex from step (1) into the cell using a knowndelivery means. Alternatively, the method of the invention may comprise(1) admixing a transfection agent with a nucleic acid and (2)introducing a peptide or protein, optionally covalently conjugated to anucleic acid binding group. Additionally, both methods may be combinedusing any number of proteins or peptides. One of ordinary skill in theart, based on knowledge generally available to the art including thepresent disclosure, can use the compositions and peptides or proteins ofthe present invention with any delivery system without the expense ofundue experimentation.

[0106] This invention includes pharmaceutical compositions, therapeuticcompositions and diagnostic compositions. In each case thesecompositions comprise an amount of transfection composition of thisinvention sufficient for effecting introduction of a selected nucleicacid into a target cell or target tissue. Pharmaceutical and therapeuticcompositions of this invention comprise suitable pharmaceuticalcarriers. Any cationic or polycationic species in these pharmaceuticalor therapeutic compositions can be provided as salts withpharmaceutically appropriate counter ions.

[0107] Kits comprising components of the transfection compositions ofthis invention can be employed to facilitate preparation and use oftransfection compositions. Such kits can be provided and employed asresearch reagents for any transfection of eukaryotic cells done forresearch purposes. Such kit may also be used for diagnostic andtherapeutic applications. Kits can be configured with componentsadequate for use in single transfection or for multiple transfections.In one embodiment, kit components comprise a cationic lipid compositionand a peptide or protein or modified-peptide or protein to enhancetransfection. The cationic lipid composition comprises a cationic lipidand preferably a neutral lipid. Preferred cationic lipid compositionscomprise a monocationic or polycationic lipid. More preferred cationiclipid compositions comprise the monocationic lipids DOTMA and DOTAP, thepolycationic lipid DOSPA, or analogs and homologs of DOSPA, includingDOSPER. Preferred neutral lipids include DOPE or DPhPE and analogs orhomologs thereof.

[0108] The level of transfection enhancement effected by a given peptideor protein or modified peptide or protein may vary dependent upon thecell type, components of the transfection agent, transfection methodused, the order of addition of components to or the order ofcomplexation of components in a transfection composition, among otherfactors. Those of ordinary skill in the art using the guidance andmethods provided herein and with knowledge of procedures, assays andmethods for transfection well-known in the art can select, without undueexperimentation, a particular peptide or protein or modified peptide orprotein of this invention for enhancement of transfection in a givensystem.

[0109] It will be readily apparent to those of ordinary skill in the artthat a number of parameters are important for optimal transfection. Forcationic lipid-mediated transfection, these parameters include cationiclipid concentration, relative amounts of cationic and non-cationiclipid, the concentration of nucleic acid, the medium employed fortransfection, the length of time the cells are incubated withtransfection composition, the amount of peptide employed, the amount ofDNA-binding group or polyamine employed, and the way, e.g., order, inwhich the components of the transfection composition are combined. Fordendrimer-mediated transfection, these parameters include dendrimersize, shape and chemical composition, the relative amount of dendrimerand nucleic acid, the addition of other transfection agents(DEAE-dextran, chloroquine),the concentration of nucleic acid, themedium employed for transfection, the length of time the cells areincubated with transfection composition, the amount of peptide employed,the amount of DNA-binding group or polyamine employed, and the way(e.g., order) in which the components of the transfection compositionare combined. It may be necessary to optimize these parameters for eachcell type (for each kind of transfection system) to be transfected. Suchoptimization is routine employing the guidance provided herein andtransfection assays as described in the Examples herein.

[0110] It will also be apparent to those of ordinary skill in the artthat alternative methods, reagents, procedures and techniques other thanthose specifically detailed herein can be employed or readily adapted toproduce the transfection compositions of this invention and practice thetransfection methods of this invention. Such alternative methods,reagents, procedures and techniques are within the spirit and scope ofthis invention.

[0111] The transfection compositions and methods of this invention arefurther illustrated in the following non-limiting Examples. Allabbreviations used herein are standard abbreviations in the art.Specific procedures not described in detail in the Examples arewell-known in the art.

[0112] All publications and patents referred to herein are specificallyincorporated by reference in their entirety.

EXAMPLES Example 1 Peptides and Peptide Conjugates

[0113] Peptides were synthesized using automated solid phase peptidesynthesis as described, e.g., in Stewart et al. (1984) Solid PhasePeptide Synthesis, Pierce Chemical Company, Rockford, Ill. Peptides weresynthesized using a polyamide-Kieselguhr composite resin and a Milligen9050 peptide synthesizer (Milligen/Biosearch, Burlington, Mass.).Coupling cycles were performed according to the manufacturer'srecommendations. 9-Fluorenyl-methyloxy-carbonyl (Fmoc) amino acid wasactivated as a pentafluorophenyl ester (—OPfp ester); Peptides weredeblocked using; (1) 20% piperidine in N,N-dimethylformamide (DMF) foralpha-amino groups; Peptides were cleaved from the resin and deprotectedusing 95% trifluoroacetic acid (TFA), (2) Reagent R [TFA (90%),thioanisol (5%), ethylene dithiole (3%) and anisole (2%)], Reagent B[TFA (88%), phenol (5%), triisopropylsilane (2%) and water (5%)] orReagent T [TFA (95%), triisopropylsilane (5%); Deprotection agent beingchosen as is understood in the art based on the protecting groups usedand the type of amino acid residues in the peptide; Crude peptides wereprecipitated and washed with ether. Peptides were purified by highpressure liquid chromatography on a Vydac C-18 reverse-phase columnusing a Waters HPLC system. The mobile phase consisted of a gradientfrom 0.01% TFA in 95% water/acetonitrile to 0.01% TFA in 25%water/acetonitrile. Peptides were characterized by HPLC, amino acidanalysis and mass spectrometry (ES or MALDI-TOF). Exemplary peptidesequences useful in this invention are listed in Tables 1-3.

[0114] It was found that certain peptides that were only partiallydeblocked, i.e., at least one amino acid protecting group had not beenremoved, showed significant enhancement of transfection. Appropriatechoice of deprotection agent allows selective synthesis of peptideswhich retain a desired protecting group. For example, deprotection withReagent T does not remove the Mtr protecting group on arginines allowingthe synthesis of partially deblocked peptides with Mtr groups remainingon R residues.

[0115] Synthesis of Polyamine Conjugated Peptides

[0116] Peptides can be modified with polyamines, such as spermine, usingan automated peptide synthesizer. Either the Fmoc or Boc chemistries canbe used. For example, spermine can be attached to the N-terminus of apeptide as illustrated in Scheme I. 5-Carboxy spermine and Boc-protected5-carboxy spermine can be synthesized as described in Behr, J- P. et al.(1989) Proc. Natl. Acad. Sci., 86:6982-6986. Fmoc-carboxy spermine canbe synthesized by treating carboxy spermine with 9-fluorenylmethylchloroformate. Fmoc-carboxy spermine or the pentafluorophenyl ester canbe used in the synthesizer to obtain spermine- modified peptides. Morethan one polyamine can be attached in this manner to a given peptideusing an appropriate combination of protecting groups.

[0117] N^(I), N^(II), N^(III),N^(IV)-tetra(9-fluorenylmethoxycarbonyl)-5-carboxyspermine(Fmoc-carboxyspermine)

[0118] 5-Carboxy spermine (11.0 g) was dissolved in 100 ml water. Thesolution was chilled on ice and diluted with 200 ml dioxane and flushedwith argon. A solution of 50 g of 9-fluorenylmethyl chloroformate(Fmoc-Cl) in 200 ml of dioxane was slowly added to the chilled carboxyspermine. The reaction mixture was stirred under argon at 4° C. for anhour and at room temperature overnight under argon. The reaction wasmonitored by TLC (silica Gel, CHCl₃/MeOH :: 9/1). The reaction mixturewas poured into 1.5 L ice cold water (1.5 L) and extracted with ethylacetate (2 L). The organic layer was separated and sequentiallyextracted with 400 ml of 1 N HCl (2×) and 300 ml saturated NaCl. Theorganic layer was dried over Na₂SO₄ and concentrated on a rotaryevaporator. The resultant gummy material was subjected to flashchromatography (silica, CHCl₃/MeOH:: 95/5) to yield 25.5 g of thedesired material as a white fluffy solid.

[0119] Fmoc-carboxy-spermine-OPfp Ester

[0120] A solution of pentafluorophenol (4.2 g) in 3 ml of dioxanesolution, followed by 2 ml of dioxane, was added to a solution ofFmoc-carboxy-spermine (8.0 g) in 10 ml of dioxane. The resulting mixturewas chilled in an ice water bath and flushed with argon. Freshlydistilled dicyclohexylcarbodiimide (DCC, 1.6 g) in 4 ml of dioxane wasadded to the chilled reaction mixture. The mixture was stirred at 4° C.for about an hour and at room temperature overnight under argon.Precipitated dicyclohexyl urea (DCU) was filtered out of the reactionmixture, which was then concentrated to dryness on a rotary evaporator.The residue was dried under high vacuum overnight. The ester wasobtained in quantitative yield as a slightly yellowish gum, which wasused without further purification in the peptide synthesizer.

[0121] Synthesis of Spermine-Modified Peptides

[0122] Spermine-modified peptides were synthesized using Fmoc chemistryon a Milligen 9050 synthesizer using the protocol suggested by themanufacturer. Peptides were synthesized conventionally andcarboxyspermine was attached at the N-terminus of the synthesizedpeptide using Fmoc-carboxyspermine-OPfp ester as the last amino acid tobe added on the synthesizer, as illustrated in Scheme 1. Deprotectionreagents used were selected as discussed above. The peptide-spermineconjugates were stored frozen until use. Table 4 lists several examplesof spermine-conjugated peptides that were synthesized usingFMOC-carboxy-spermine.Modified peptides were analyzed and purified usingHPLC on a Vydac C-18 column. Modified peptides were characterized byHPLC, amino acid analysis and mass spectrometry (ES or MALDI-TOF). Thismethod can be employed or readily adapted in view of well-knowntechniques for synthesis of polyamine -peptide conjugates.

[0123] Peptide-spermine conjugates can also be prepared using aheterobifunctional cross-linking agent sulfo-SMPB (Pierce Chemical Co.,Rockford, Ill.) as illustrated in Scheme II. See: S. S. Wong“Heterobifunctional Cross-linkers” in Chemistry of Protein Conjugationand Cross-linking CRC Press p.147-194. Briefly, 100 mg/mL sulfo-SMPB inDMF is diluted to 20 mg/mL using 50 mM sodium phosphate buffer (pH 7.5).Spermine (50 mg/mL in 50 mM sodium phosphate buffer) was then added tothe sulfo-SMPB solution at a 3:1 molar ratio. After 1 hour at roomtemperature, the reaction mixture is fractionated (LH-20 column) usingthe sodium phosphate buffer. The first major peak (spermine-MPB) wascollected. Spermine-MPB is mixed at a 1:1.5 to 1:2 ratio with asynthetic (or naturally-occurring) peptide with terminal cysteine (HS—),either in pure powder form or in acetonitrile/water solution. Excesspeptide is separated on a LH-20 column eluted with water. Thepeptide-spermine conjugate is stored frozen until use. The reaction ofspermine-MPB with HS-peptide should be performed under appropriatereducing conditions to avoid peptide dimer formation. This method can beemployed or readily adapted in view of well-known techniques forsynthesis of polyamine -peptide conjugates.

[0124] Peptide dimers of peptides containing cysteine residues can beformed, if desired, by oxidative coupling to form a disulfide bondbetween two peptides. Concatemers and mixed concatemers of thisinvention can be prepared by automated peptide synthesis and if desiredthe concatemers, mixed concatemers and peptide oligomers (dimers, etc.)can be conjugated to nucleic acid-binding groups by methods describedherein.

[0125] Scheme 3 illustrates appropriately protected polyamine speciesthat can be used in automated peptide synthesizers to introducepolyamines at the carboxy terminus of peptides (e.g., Structure II, forcarboxyspermine conjugation). The scheme illustrates a synthesis of thecarboxyspermine derivative of Structure II, which can be readilygeneralized for the synthesis of any analogous polyamine derivatives.The compound of Structure II, activated by removal of thepentafluorophenol group, can be conjugated to the solid support toprovide a carboxy spermine group at a peptide carboxy terminus. Standardautomated peptide synthesis is performed using the support of StructureI. The fully protected carboxy spermine of Structure II can itself beemployed as a reagent for addition of the carboxy spermine group at anyposition along a synthetic peptide chain. These methods can be readilyand routinely adapted for conjugation of any polyamine.

[0126] Peptide-Lipid Conjugates

[0127] Peptide-lipid conjugates are prepared as follows: A lipid with areactive head group, for example a group such as1,2-dioleoyl-S,N-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate](N-PDP-PE, Avanti Polar Lipids, Inc. Alabaster, Ala.) is reacted with acysteine-peptide (e.g.,cysteine at either end of the functionalpeptide). Reaction can be performed at RT and followed by measuring thereleased chromophore. See Scheme 4. For example, N-PDP-PE is dissolvedin methanol to a concentration of 0.2 M (about 200 mg/mL). Cys-peptides,including VSVG-Cys, (e.g., KFTIVFC, SEQ ID NO:11); RGD—Cys (e.g.,GRGDSPC, SEQ ID NO:8); or Cys-NLS (e.g., CGWGPKKKRKVG, SEQ ID NO 12),are dissolved in an appropriate solvent, e.g., DMF, to a concentrationof 100 mg/mL and mixed with the N-PDP-PE solution at a molar ratio of1.5-2: 1. Peptide-lipid conjugates can be purified by HPLC using a Vydacprotein and peptide C 18 reverse phase column with anacetonitrile/water/TFA and methanol solvent system. The conjugate can becharacterized by UV and MS analysis.

Example 2 Enhancement of Cationic Lipid Transfection of Human FibroblastCells With Viral Peptides Added into Transfection Medium.

[0128] The following viral peptides were synthesized using automatedsolid phase peptide synthesis as described in Example 1: the membranefusion region of influenza virus (HApep)(see, Epand et al. (1992)Biopolymers 32:309); modifications of FluHa to yield hemagglutininpeptides E5 and K5 (see, Kamata, H. et al. (1994) Nucleic Acids Res.22:536-537); and vesicular stomatitis virus G-protein, VSVG (see,Schlegel, R. and Wade, M. (1985) J. Virol. 53:319). The nuclearlocalization signal (NLS) of SV40 large T antigen, NLS (see, Lanford etal. (1986) Cell 46:575) and the RGD peptide (Ruoslahti, E. andPierschbacher, D. (1987) Science 238:491) were also synthesized. Thesequences of the peptides synthesized is given in Table 5.

[0129] Newborn human fibroblasts (NHF) were isolated from neonatalforeskin dermis and prepared as described in, Hawley-Nelson, P., et al.(1993) Focus 15:73, incorporated by reference herein, and cultured forup to 20 passages. Cultures of adherent cells were grown inDulbecco's-modified Eagle's medium (DMEM) containing 0.1 mM MEMNon-Essential Amino Acids (NEAA), 10% (v/v) fetal bovine serum (FBS),100 U/mL penicillin (PEN) and 100 μg/mL streptomycin (STREP). Cultureswere passaged at confluence using 0.25% (v/v) trypsin, 0.1 mM EDTA.

[0130] The plasmid vectors pCMV13gal and pCMVSPORTpgal are commerciallyavailable (Clontech, CA and GIBCO-BRL, respectively) mammalian reportervectors containing the E. coli B-galactosidase (13-gal) gene under thecontrol of the Cytomegalovirus promoter. See: MacGregor et al. (1989)Nucleic Acids Res. 17: 2365; Norton et al. (1985) Mol. and Cell Biol.5:281; Alam (1990) Anal. Biochem. 188:245. Plasmid DNA was purified bystandard cesium chloride methods.

[0131] Human fibroblasts were plated the day before transfection at8×10⁴ per well on a 24-well dish. Before transfection, the cells wererinsed with serum-free DMEM. Two 25 μl aliquots of “OPTI-MEM”-I medium,one containing 3 μg “LIPOFECTAMINE” and the other containing 0.2 μgpCMVβgal DNA, were combined to form complexes for 30 min at roomtemperature. “LIPOFECTAMINE” (Gibco/BRL: Life Technologies, Inc.,Gaithersburg, Md.) is a 3:1 (w/w) mixture of the polycationic lipid,2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), and DOPE. Peptides were dissolved indimethylsulfoxide (DMSO) at 250× the final concentration (see Table 5).Peptide solution (1 μL) was added to 250 μl serum-free DMEM transfectionmedium and added to the rinsed cells. Treatments containing the E5, K5,HApep, and VSVG alone, and E5+K5 and VSVG+K5 in combination with eachother were compared to a transfection sample containing no peptide(“LIPOFECTAMINE”+DNA only). For treatments that combined two peptides,E5, K5 and VSVG were all used at 5 μM concentrations. The DNA-lipidaggregates in “OPTI-MEM”-I were then added to the transfection mediumwith added peptide(s) on the cells. After 24 hours incubation at 37° C.,cells were harvested, extracted and assayed for β-galactosidase activityas described above.

[0132] Enzyme activity of lysed cell extracts was used to compare levelsof expression resulting from different treatment protocols. One to twodays following transfection, cells were rinsed once with PBS and frozenat −70° C. in 0.15 mL/well 0.1% “TRITON” X-100(t-octylphenoxypolyethoxyethanol; Sigma Chemical Co., St. Louis, Mo.;“TRITON” is a trademark of Union Carbide, Inc.) and 0.1M Tris, pH 8.0.After rapid thawing at 37° C., the lysate was cleared by centrifugation.Lysed cell extracts were assayed for 13-galactosidase activity employingthe method essentially as described in Sambrook et al. (1989) MolecularCloning A Laboratory Manual, 2^(nd) Ed., Cold Spring Harbor LaboratoryPress, p. 16.66. Briefly, soluble cell extract containing 2-6 μg proteinwas added to 100 μl 0.1M sodium phosphate buffer (pH 7.5) containing 1mM MgCl₂, 50 mM 13-mercaptoethanol and 0.88 mg/mLo-nitrophenyl-β-D-galacatopyranoside (ONPG) in a 96-well microtiterplate. A standard curve of 10-70 ng β-galactosidase (Gibco/BRL: LifeTechnologies, Inc., Gaithersburg, Md.) was included on the plate. Yellowcolor developed in 5-20 minutes at 37° C. The reaction was stopped whennecessary by adding 150 μl 1 M Na₃CO₂; OD₄₂₀ was determined on amicrotiter plate reader.

[0133]FIG. 1 shows the effect of peptides added into the DMEMtransfection medium after lipid has been complexed to DNA. As shown inFIG. 1, relatively minor enhancements of transfection compared to thecontrol were observed, except for HApep and the combination of VSVG andE5. The VSVG+E5 combination showed greater than 2-fold enhancementcompared to “LIPOFECTAMINE” alone. Peptide concentrations that resultedin optimal transfection are listed in Table 5. It was later found thatthe order of addition of components had a significant effect upontransfection enhancement by peptides. Initial complexation of thepeptide with the nucleic acid prior to contact of the DNA with thecationic lipid composition in general gave significantly highertransfection enhancement.

Example 3 Transfection Enhancement of Sp-NLSNLS Pre-complexed to DNA inCombination with “LIPOFECTAMINE”

[0134] Sp-NLSNLS (the peptide (NLSNLS): GYGPKKKRKVGGGGYGPKKKRKVGG [SEQID NO:13] conjugated to spermine) enhances transfection efficiency incombination with “LIPOFECTAMINE” in human fibroblasts, NIH 3T3, MDCK andBHK-21 cells when the peptide is pre-complexed to the DNA prior toaddition of “LIPOFECTAMINE.”

[0135] In this example, all media, sera, and reagents were from GIBCOBRL unless otherwise noted. All cells were cultured in Dulbecco's MEM(DMEM, high glucose: 4,500 mg/L D-glucose, with L-glutamine and phenolred) with 0.1 mM Non-Essential Amino Acids (NEAA), 100 U/mL penicillinand 100 μg/mL streptomycin. Human fibroblasts, MDCK, and BHK-21 cellswere cultured with 10% Fetal Bovine Serum (FBS). NIH 3T3 cells werecultured with 10% Calf Serum. Human fibroblast cells were obtained asdescribed in Example 2. NIH 3T3 (NIH Swiss mouse embryo,contact-inhibited fibroblasts), MDCK (dog kidney cells) and baby hamsterkidney (BHK-21) cells were obtained from the American Type CultureCollection (Rockville, Md.). All cultures were maintained at 37° C. with5% CO₂. Human fibroblasts at 6×10⁴, BHK-21 at 4×10⁴, MDCK at 6×10⁴, andNIH 3T3 at 5×10⁴ cells per well were plated the day before transfectionin 24-well plates. On transfection day, the cell cultures were 50-80%confluent.

[0136] For each well on a 24-well plate, 0.5-4 μL “LIPOFECTAMINE”Reagent and 0.1-0.8 μg pCMV·SPORT-β-gal or pCMVβ DNA (G. R. McGregor andC. T. Caskey (1989) Nucleic Acids Res. 17:2365) were diluted intoseparate 25 μL aliquots of serum-free medium (“OPTIMEM-1” or D-MEM withadded 0.1 mM NEAA). Sp-NLSNLS (1-4 μg, as a 1 mg/ml solution in water)was added into the DNA solution, and incubated at RT for 15 min to allowpre-complexation. The DNA-Sp-NLSNLS solution was mixed together withdiluted “LIPOFECTAMINE”, then incubated at RT for 30 min. The cells wererinsed with serum-free DMEM with NEAA, then 0.2 ml per well ofserum-free DMEM with NEAA was added to the cells. TheDNA-Sp-NLSNLS-lipid complex was added to the serum-free D-MEM on thecells, incubated at 37° C. for 5 hrs, then 1 ml of D-MEM containing FBS(final concentration 10%) was added. Cultures were incubated 24 hrs,then fixed and stained in-situ with X-gal (J. R. Sanes et al. (1986)EMBO J. 5:3133, see below) or harvested for ONPG assay (as described inExample 2).

[0137] In order to test enhancement of transfection efficiency, humanfibroblasts were transfected with pCMV-SPORT—O-gal DNA using“LIPOFECTAMINE” and Sp-NLSNLS. Increasing concentrations of Sp-NLSNLSwere tested and β-galactosidase activity was assayed with ONPG (FIG. 2).In this transfection, 2-20 μg Sp-NLSNLS precomplexed with 0.4 μg DNAresulted in a plateau of enhanced activity. Enhancement levels between5-8-fold were routinely observed at 2-4 μg Sp-NLSNLS per 0.4 μg DNA insimilar experiments (data not shown).

[0138] Human fibroblasts similarly transfected were stained in-situ withX-gal. The results of the most active concentration of Sp-NLSNLS (2 μg)and lipid (2 μL) with DNA (0.4 μg) was photographed and positive cellscounted. Percent positive cells (mean of 3 determinations +/−half therange) was determined: “LIPOFECTAMINE” (4+/−2%), “LIPOFECTAMINE” withSp-NLSNLS (18+/−6%). This is a four-fold enhancement in percenttransfected cells.

[0139] In situ staining was used to demonstrate 13-galactosidaseexpression. Cells were rinsed with PBS, fixed for 5 min in 2% (v/v)formaldehyde, 0.2% glutaraldehyde in PBS, rinsed twice with PBS, andstained overnight with 0.1% X-gal (Gibco/BRL: Life Technologies, Inc.,Gaithersburg, Md.), 5 mM potassium ferrocyanide, 5 mM potassiumferricyanide, 2 mM MgCl₂ in PBS. Rinsed cells were photographed using a10× objective on a Nikon inverted microscope with Hoffman optics.Transfection efficiency was evaluated by counting or estimating thenumber of β-gal positive (blue-stained) cells.

[0140] Analyses of transfection enhancement by Sp-NLSNLS were performedin human fibroblasts, BHK-21, NIH3T3, and MDCK cells using wide rangesof lipid and DNA concentrations. The data are presented in FIGS. 3A-3H.In each case, the whole platform of activity is raised, not just theactivity at the optimum of lipid and DNA concentrations. Enhancement ofthis type facilitates the reproducibility of high efficiencytransfections, broadening the spectrum of activity of polycationiclipid-mediated transfection (particularly transfection using“LIPOFECTAMINE” and resulting in high activity over a broader range ofDNA and lipid concentrations. The use of spermine-derivatized peptides,such as Sp-NLSNLS, in transfections decreases the amount of detailedoptimization of lipid and DNA concentration previously required toachieve high activity cationic lipid transfections in a given system.

[0141] By increasing the platform of activity across the ranges of lipidand DNA concentrations with Sp-NLSNLS and other peptides andspermine-derivatized peptides, it is possible to achieve high leveltransfections with lower amounts of lipid and DNA resulting in highercell yield from equivalent or higher efficiency transfection.

[0142] Table 6 illustrates this observation using data generated in theexperiment graphed in FIGS. 3A and 3B. Peak activity of 0.07 ng β-gal/μgprotein is achieved with “LIPOFECTAMINE” alone at 0.4 μg DNA and 2 μL“LIPOFECTAMINE”. The protein yield (which is directly proportional tocell yield) for these conditions was 67.5 μg/well. By using Sp-NLSNLS0.38 ng β-gal activity per μg protein (over 5-fold higher than the peakfor “LIPOFECTAMINE” alone) can be achieved in a transfection with 0.1 μgDNA and 1 μl “LIPOFECTAMINE.” The protein yield under these conditionsis 85.5 μg/well, 25% more than at the peak with “LIPOFECTAMINE” alone.This difference is seen in other cell types, as well (data not shown).The enhanced yield is most likely due to the use of lower lipid and DNAconcentrations, since similar yields are seen with “LIPOFECTAMINE” aloneand with Sp-NLSNLS-precomplexed DNA at the same lipid and DNAconcentrations (data not shown).

Example 4 Enhancement of “LIPOFECTAMINE” Transfection in HumanFibroblast Cells by Peptides and Peptide Derivatives Pre-Complexed toDNA.

[0143] Table 7 compares transfection activity for “LIPOFECTAMINE”combined with various peptides and peptide derivatives. Transfectionswere done in human fibroblasts (except as indicated) using pCMSPORTβgalor pCMVβ using the ONPG assay as described in Example 3. Experimentswere done in 24-well plates using 0.4 μg DNA/transfection (except asindicated). The protocol described in Example 3 was used with thepeptides added to DNA initially. The data in Table 7 are thefold-enhancement at peak activity for “LIPOFECTAMINE” and the peptide ormodified peptide. During the purification of the peptides by HPLC inseveral instances, two peaks were isolated. K16NLS (peak 2) isincompletely deprotected material which is believed to retain theMtr-protecting group on an arginine residue (R). Most preferredtransfection enhancing agents are those peptides or peptide derivativesthat give the highest fold enhancement (compared to cationic lipidalone) at the lowest amount of enhancing agent. Note that the reverseNLS peptide exhibited no enhancement of transfection with“LIPOFECTAMINE”.

Example 5 Transfection Activity of DMRIE-C in the Presence of CertainPeptide Derivatives

[0144] Table 8 compares transfection activity for DMRIE-C combined withseveral different peptide (or peptide derivatives or combinationsthereof ) for transfection of suspension cell lines (K562 and Jurkatcells).

[0145] DMRIE-C is a 1:1 (M/M) liposome formulation with cation lipidDMRIE (1,2-dimyristyloxypropyl-1-3-dimethylhydroxyethyl ammoniumbromide) and cholesterol in membrane-filtered water. See: K. Schifferliand V. Ciccarone (1996) “FOCUS” 18:45 and V. Ciccarone et al. (1995)“FOCUS” 17:84. Transfections were performed using pCMVSPORTCAT, and theassays were performed using CAT assay: varying amounts of peptidederivatives (or mixtures of such derivatives) were combined andpre-incubated with DNA for 15 minutes. The precomplexed DNA-peptide (andor peptide derivative) complex was then mixed with 1.6 μL DMRIE-C. Thetransfection composition was then mixed with 4×10⁵ cells/well in 24-wellplates. CAT assays were performed at 36-48 h after transfection.

[0146] The chloramphenicol acetyltransferase (CAT) assay was preformedas described in J. R. Neuman et al. (1987) BioTechniques 5:444. Briefly,harvested cells from a well were washed with PBS and pelleted bycentrifugation at 1000 rpm (˜600× G) for 5 m at RT for suspension cells.Pellets were put on ice and 1 mL of 0. 1M Tris-HCl (pH 8.0) containing0.1% TRITON X-100 was added and then frozen at −70° C. for 2 h. Pelletsare thawed at 37° C., then chilled on ice. Cell lysates were centrifugedat maximum speed in a microcentifuge for 5 min. Supernatant wascollected and heated for 10 m at 65° C. to inactivate deacetylases andother inhibitors of the CAT reaction. Heated supernatants (hereaftercell extracts) were centrifuged at maximum speed for 3 m and stored at−70° C.

[0147] For each cell extract sample, add 5-150 μL cell extract and makeup to 150 μL with 0.1 M Tris-HCl (pH 8.0). Negative control is 150 μL0.1 M Tris-HCl (pH 8.0); Positive control is CAT standard solution (1,5, 10, 20, and 50 mU of CAT) made up to 150 μL with 0.1 M Tris-HCl (pH8.0). Add 100 μL of a mixture: 10 μL 1 M Tris-HCl (pH 8.0); 1 μL 250 mMchloramphenicol (in 100% ethanol); 5 μL (50nCi) [1⁴C-butryl Coenzyme A(0.010 μCi/μL); 84 μL deionized, distilled water, to each sample andincubate at 37° C. for 2 h. Add 3 mL of “ECONOFLUOR” to each sample andincubate at RT for 2 h. Count each sample for 0.5 m in a liquidscintillation counter.

[0148] Table 8 lists the highest fold enhancement observed with peptideused and the amount of peptide (or derivative) needed to achieve thatlevel of enhancement.

Example 6 Enhancement of Dendrimer-Mediated Transfection

[0149] “STARBURST” polyamidoamine (PAMAM) dendrimers: G7 (EDA), G9(EDA),and G6(EDA) modified with lysine [Lys DMER] or with arginine [Arg DMER],and a “COMB BURST” (Trademark, Dendritech Inc.) dendrigraft wereobtained from Michigan Molecular Institute. The PAMAM dendrimers wereprepared by now standard methods as described, for example, in TomaliaD. A. and Durst, H. D. (1993) in Weber E. (ed.) Topics in CurrentChemistry, 165: “Supramolecular Chemistry I-Directed Synthesis andMolecular Recognition, Springer-Verlag, Berlin pp. 193-313 and TomaliaD. A. et al. (1990) Angew. Chem. Intl. Ed. Engl. 29:138-175. Thesemodified dendrimers were stored as the trifluoroacetate salts. Lys DMERhas a charge density of 2.07×10¹⁵+charge/μg; Arg DMER has a chargedensity of 2.41×10¹⁵+charge/μg. “COMB BURST” dendrigraft was grown togeneration three and then modified with one layer of PAMAM repeat unitsto give a polymer with a molecular weight of 30,000, a polydispersity of1.11 and a charge density of 2.68×10¹⁵+charges/μg.

[0150] The effect of peptides and spermine-peptide conjugates ondendrimer-mediated transfection was assessed in transfections of COS-7cells (ATCC). Dendrimer transfection was performed essentially asdescribed in Kukowska-Latallo J. F. et al. (1996) Proc. Natl. Acad. Sci.USA 93::4897-4902. COS-7 cells were plated at 4×10⁴ cells/well in 24well plates. Two DNA plasmids were used: pCMVβ for X-gal staining andpGL3 (Promega) for luciferase assay both at 0.5 μg/well. All dendrimerswere used at 3 μg/well and chloroquine was added to all dendrimertransfections at 25 μg/mL. Dendrimer transfections were compared with“LIPOFECTAMINE” transfections using 1 μL/well. The effect of threepeptides: K16NLS (peak 2, incompletely deblocked) added at 1 μg/well,Sp-NLSNLS added at 1.5 μg/well, and NLS added at 20 μg/well wasdetermined.

[0151] For the luciferase assay, each well was extracted in 0.15 mllysis buffer (25 mM Tris HCl, pH 8.0, 0.1 mM EDTA, 10% glycerol, 0.1%Triton X 100), and 10 μL centrifuged extract supernate from each wellwas automatically mixed with 50 μL Luciferase Assay Reagent (Promega)and assayed in a luminometer for 5 seconds. The X-gal assay wasperformed as in Example 3.

[0152] The results of the luciferase assay are listed in Table 9. Thetransfection activity of each of the dendrimers in Table 11 was enhancedby the three peptides or peptide conjugates tested. The most activedendrimers for transfection of COS-7 cells were Arg DMER and “COMBBURST”. In these experiments, transfection was not optimized fordendrimer concentration or the amount of peptide added. X-gal assayswere consistent with the data given in Table 9.

Example 7 Effect of Sp-NLSNLS on Stable Transformation Frequency in NIH3T3 Cells

[0153] NIH 3T3 cells were seeded at 6×10⁴ cells/well in 24 well plates,in DMEM supplemented with 10% Calf Serum, and allowed to grow overnight.Cells were transfected in matrix format using 0, 1, 2, 3, and 4 μL“LIPOFECTAMINE”, and 0.1, 0.2, 0.4, and 0.8 μg pSV2neo (obtained fromATCC, carrying a neomycin resistance gene, see: Berg et al. (1982) J.Mol. Appl. Genet. 1: 327-341. DNA was pre-incubated with 2 μg (for0.1-0.4 μg DNA) or 4 μg (for 0.8 μg DNA) peptide. Control DNA wasprovided with no peptide (here Sp-NLSNLS, see Table 5 for sequence). At24 h after transfection, cells were split 1:150 into 35 mm platescontaining growth medium, and allowed to grow overnight. The next daycells were put in selection medium containing 0.6 mg/ml “GENETICIN”(G418 sulfate, Gibco/BRL: Life Technologies, Inc., Gaithersburg, Md.)).After 10 days plates were fixed and stained with 10% formalin/PBScontaining 0.4% toluidine blue. G418-resistant colonies were counted andexpressed as % of initial number of cells plated. FIGS. 4A (no peptidecontrol) and 4B (with added Sp-NLSNLS) show the results of thisexperiment. Significant enhancement of transfection by Sp-NLSNLS isobserved in almost all cases.

Example 8 Effect of Sp-NLSNLS on Transfection by Monocationic LipidReagents

[0154] In separate experiments, BHK-21 cells were seeded at 2×10⁴cells/well, COS-7 cells were seeded at 5×10⁴ cells/well, CHO-KI cellswere seeded at 6×10⁴ cells/well, human fibroblasts were seeded at 8×10⁴cells/well, and HT29 cells were seeded at 1×10⁵ cells/well in 24-wellplates in DMEM supplemented with 10% fetal bovine serum and allowed togrow overnight. Cells were transfected as described in example 3, using0-4 ml “LIPOFECTIN”, “LIPOFECTACE” (1:2.5 w:w ratio ofdimethyl-dioctadecyl-ammonium bromide (DDAB) and DOPE), or DMRIE-DOPE (1:1 molar ratio, 1 mg/ml) and 0.4 mg pCMVSPORTbgal plasmid DNAprecomplexed with 0-15 mg Sp-NLSNLS peptide. At 24 h after transfection,cells were harvested as described in example 2. Lysates were assayed forb-galactosidase activity using the luminescent assay from Tropix (V. K.Jain and I. T. Magrath (1991) Anal. Biochem. 199:119-124). Table 10shows the peak activities of transfection with a range of concentrationsof the monocationic lipid with DNA alone or with DNA precomplexed toSp-NLSNLS peptide. A clear enhancement is seen in most cases.

Example 9 Effect of Sp-NLSNLS on transfection by polycationic lipidreagents other than DOSPA-DOPE

[0155] In separate experiments, CHO-KI and NIH3T3 cells were seeded at6×10⁴ cells/well, and human fibroblasts were seeded at 8×10⁴ cells/wellin 24-well plates in DMEM supplemented with 10% fetal bovine serum (10%calf serum for NIH3T3) and allowed to grow overnight. Cells weretransfected as described in example 3, using 0-4 ml “CELLFECTIN,” TMDOS,“DOSPER” or “MULTIFECTOR” and 0.4 mg pCMVSPORTbgal plasmid DNAprecomplexed with 0-40 mg Sp-NLSNLS peptide. At 24 h after transfection,cells were harvested as described in example 2. Lysates were assayed forb-galactosidase activity using ONPG (see Example 2) or the luminescentassay from Tropix (see Example 8). Table 11 shows the peak activities oftransfection with a range of concentrations of the polycationic lipidswith DNA alone or with DNA precomplexed to Sp-NLSNLS peptide. A clearenhancement is seen.

Example 10 Effect of Sp-NLSNLS on transfection by the activateddendrimer “SUPERFECT”

[0156] COS-7 cells were seeded at 4×10⁴ cells/well in 24-well plates inDMEM supplemented with 10% fetal bovine serum and allowed to growovernight. Cells were transfected as described in example 3, using 0-4ml “SUPERFECT” and 0.4 mg pCMVSPORTbgal plasmid DNA precomplexed with0-5 mg Sp-NLSNLS peptide. At 24 h after transfection, cells wereharvested as described in Example 2. Lysates were assayed forb-galactosidase activity using the luminescent assay from Tropix (seeExample 8). Table 12 shows the peak activities of transfection with arange of concentrations of the dendrimers with DNA alone or with DNAprecomplexed to Sp-NLSNLS peptide. A clear enhancement is seen.

Example 11 Effect of TAT and TAT-Spermine Peptides on Transfection by“LIPOFECTAMINE”

[0157] Two versions of the Tat peptide were synthesized: one with theDNA-binding group carboxyspermine on the N-terminus and one without. Thecore TAT sequence employed was CGYGRKKRRQRRRG. The ability of the TATpeptide and the carboxyspermine-modified TAT peptide to enhance cationiclipid-mediated transfection was tested.

[0158] Transfection was performed in NIH/3T3 cells, in 24-well plates.Seeding density was 6×10⁴ cells/well. For each condition, the standardPLUS protocol was used (see example 3) for complexing. All complexeswere prepared in OptiMEM reduced serum media. Cells were harvested andassayed for β-galactosidase acivity in the soluble extracts byluminescent assay (see example 8). Table 13 shows the peak activities oftransfection with a range of concentrations of “LIPOFECTAMINE” with DNAalone or precomplexed to the Tat or Tat-spermine peptides. A clearenhancement is seen.

Example 12 Effect of Including Receptor-Ligand Proteins with Sp-NLSNLSin DNA Pre-Complexes on Transfection by “LIPOFECTAMINE” and “LIPOFECTIN”

[0159] In separate experiments, CHO-K1 cells were seeded at 6×10⁴cells/well, and human fibroblasts were seeded at 8×10⁴ cells/well in24-well plates in DMEM supplemented with 10% fetal bovine serum andallowed to grow overnight. Cells were transfected as described inExample 3, using 0-5 μl “LIPOFECTAMINE,” “LIPOFECTIN,” or “DMRIE-DOPE”and 0.4 μg pCMVSPORTβgal plasmid DNA precomplexed with 0-40 μg Sp-NLSNLSpeptide or mixtures of Sp-NLSNLS peptide and 1-2 μg Insulin or 2-4 μgTransferrin, or both 1-4 μg both Insulin and Transferrin. At 24 h aftertransfection, cells were harvested as described in Example 2. Lysateswere assayed for 6-galactosidase activity using ONPG (see Example 2) orthe luminescent assay from Tropix (see Example 8). Table 14 shows thepeak activities of transfection with a range of concentrations of thelipids with DNA alone or with DNA precomplexed to Sp-NLSNLS peptide +/−the ligand proteins. A clear enhancement is seen resulting from theinclusion of the ligand proteins in the mixture.

Example 13 Effect of an Adhesion Protein Fragment on Transfection byLipofectAMINE Reagent.

[0160] COS-7 cells were seeded at 5×10⁴ cells/well in 24-well plates inDMEM supplemented with 10% fetal bovine serum and allowed to growovernight. Cells were transfected as described in Example 3, using 0-2.4μl “LIPOFECTAMINE” and 0.8 μg pCMVSPORTpgal plasmid DNA precomplexedwith 0 or 10 μg “RETRONECTIN”. At 24 hours after transfection, cellswere harvested as described in example 2. Lysates were assayed forβ-galactosidase activity using ONPG (see Example 2). Table 15 shows thepeak activities of transfection with a range of concentrations of the“LIPOFECTAMINE” with DNA alone or with DNA precomplexed to“RETRONECTIN”. A clear enhancement is seen.

[0161] Those of ordinary skill in the art will appreciate that reagentsstarting materials, growth media, techniques and methods other thanthose specifically described herein can be employed in the preparationand use of the transfection compositions, kits, lipid aggregates,peptide and protein conjugates, modified peptides and proteins, lipids,dendrimers and peptide and protein conjugates thereof of this inventionwithout departing from the spirit and scope of this invention. TABLE 1Examples of Cell Adhesion Proteins LIGAND BINDING REGION REFERENCEFibronectin RGD cell binding region (RGDSPC) (SEQ ID NO:14)-Pierschbacher & Ruoslahti (1987) J. Biol. Chem. 262, 17294-17298 (allmotifs) Fibronectin 1 including all cell binding regions Pierschbacher &Ruoslahti (1984) Nature 309, 30-33 RGD cell binding region (all motifs)Fibronectin 2 RGD cell binding region (REDV [SEQ ID Humphries et al.,(1986) J. Cell Biol. 103, 2637-2647 NO:15]/RGDV) [SEQ ID NO:16]Fibronectin 3 CS1 Fragment [SEQ ID NO. 17]¹ Humphries et al., (1987) J.Biol. Chem. 262, 6886-6892 Vitronectin RGD cell binding region (RGDV)[SEQ ID NO:16] Suzuki et al., (1985) EMBO J. 4, 2519-2524 Laminin 3 RGDcell binding region (RGDN) [SEQ ID NO:18] Grant et al., (1989) Cell 58,933-943 Tenascin 1 RGD cell binding region (RGDM) [SEQ ID NO:19]Friedlander et al., (1988) J. Cell Biol. 107, 2329-2340 Collagen 1 RGDcell binding region (RGDT) [SEQ ID NO:20] Dedhar et al., (1988) J. CellBiol. 104, 585-593 Collagen 6 RGD cell binding region (RGDX*) [SEQ IDNO:21] Aumailley et al., (1989) Cell Res. 181, 463-474 von WillebrandRGD cell binding region (RGDS) [SEQ ID NO:22] Haverstick et al., (1985)Blood 66, 946-952 Factor Fibrinogen 1 RGD cell binding region (RGDS)[SEQ ID NO:22] Gardner and Hynes et al., (1985) Cell 42, 439-448Thrombo-spondin 1 RGD cell binding region (RGDA) [SEQ ID NO:23] Lawleret al., (1988) J. Cell Biol. 107, 2351-2361

[0162] TABLE 2 Exemplary Peptides For Enhancement of Transfection¹NLS-BASED PKKKRKV [SEQ ID NO:2] (±C)G(Y or W or -)GPKKKRKVGG [SEQ IDNO:25] C(±Y or W)PKKKRKVGG [SEQ ID NO:25] (±C)G(±Y orW)GPKKKRKVGG(±G_(n)) [SEQ ID NO:25] (Xaa)_(x)PKKKRKV(Zaa)_(z) [SEQ IDNO:24] (Xaa)_(x)(±Y or W)PKKKRKV(Zaa)_(z) [SEQ ID NO:24] (Xaa)_(x)(±Y orW)(Jaa)_(j)PKKKRKV(Zaa)_(z) [SEQ ID NO:24] (Xaa)_(x)PKKKRKV(±Y orW)(Zaa)_(z) [SEQ ID NO:24] (Xaa)_(x)(±C)(±Y orW)(Jaa)_(j)PKKKRKV(±C)(Zaa)_(z) [SEQ ID NO:24] (Xaa)_(x)(±C)(±Y orW)(Jaa)_(j)PKKKRKV(±C)(Zaa)_(z (Uaa or Sp or Poly)) _(u) [SEQ ID NO:24](±C)(Xaa)_(x)(±Y or W)(Jaa)_(j)PKKKRKV(Zaa)_(z)(±C) [SEQ ID NO:24](±C)(Xaa)_(x)(±Y or W)(Jaa)_(j)PKKKRKV(±C)(Zaa)_(z)(Uaa or Sp orPoly)_(u) [SEQ ID NO:24] Uaa or Sp or Poly)_(u)(Xaa)_(x)(±C)(±Y orW)(Jaa)_(j)PKKKRKV(Zaa)_(z)(±C) [SEQ ID NO:24] (Uaa or Sp orPoly)_(u)(Xaa)_(x)(±C)(±Y or W)(Jaa)_(j)PKKKRKV(±C)(Zaa)_(z) [SEQ IDNO:24] NLS-CONCATEMERS [(Xaa)_(x)(±C)(±Y orW)(Jaa)_(j)PKKKRKV(±C)(Zaa)_(z)]_(p) [SEQ ID NO:24] [(Xaa)_(x)(±C)(±Y orW)(Jaa)_(j)PKKKRKV(±C)(Zaa)_(z)(Uaa or Sp or Poly)_(j) [SEQ ID NO:24](Uaa or Sp or Poly)_(u)[(Xaa)_(x)(±C)(±Y orW)(Jaa)_(j)PKKKRKV(±C)(Zaa)_(z)]_(p) [SEQ ID NO:24] RGD(SP) RGDSP [SEQID NO:26] (±C)RGDSP(±C) [SEQ ID NO:26] (±C)(±G)RGDSP(±C) [SEQ ID NO:26](Xaa)_(x)RGDSP(Zaa)_(z) [SEQ ID NO:26](Xaa)_(x)(±C)(±G)RGDSP(±G)(±C)(Zaa)_(z) [SEQ ID NO:26](±C)(Xaa)_(x)(±G)RGDSP(±G)(Zaa)_(z)(±C) [SEQ ID NO:26](Xaa)_(x)RGDSPC(Zaa)_(z) [SEQ ID NO:26] (Uaa or Sp orPoly)_(u)(Xaa)_(x)RGDSP(Zaa)_(z) [SEQ ID NO:26] (Uaa or Sp orPoly)_(u)(Xaa)_(x)(±C)(±G)RGDSP(±G)(±C)(Zaa)_(z) [SEQ ID NO:26](Xaa)_(x)(±C)(±G)RGDSP(±G)(±C)(Zaa)_(z)(Uaa or Sp or Poly)_(u) [SEQ IDNO:26] (Uaa or Sp or Poly)_(u)(Xaa)_(x)(±C)(±G)RGDSP(rG)(Zaa)_(z)(±C)[SEQ ID NO:26] (±C)(Xaa)_(x)(±G)RGDSP(EG)(±C)(Zaa)_(z)(Uaa or Sp orPoly)_(u) [SEQ ID NO:26] (Xaa)_(x)RGDSP(Zaa)_(z)(Uaa or Sp or Poly)_(u)[SEQ ID NO:26] RGD(MF) RGDMF [SEQ ID NO:27] (±C)GRGDMF(±C) [SEQ IDNO:27] GRGDMFC [SEQ ID NO:27] (±C)(±G)RGDMF(±G)(±C) [SEQ ID NO:27](Xaa)_(x)RGDMF(Zaa)_(z) [SEQ ID NO:27](Xaa)_(x)(±C)(±G)RGDMF(±G)(±C)(Zaa)_(z) [SEQ ID NO:27](±C)(Xaa)_(x)RGDMF(Zaa)_(z)(±C) [SEQ ID NO:27](Xaa)_(x)(±C)RGDMF(±C)(Zaa)_(z) [SEQ ID NO:27] (Uaa or Sp orPoly)_(u)(Xaa)_(x)(±C)(±G)RGDMF(±G)(Zaa)_(z)(±C) [SEQ ID NO:27](±C)(Xaa)_(x)(±G)RGDMF(±G)(±C)(Zaa)_(z)(Uaa or Sp or Poly)_(u) [SEQ IDNO:27] (Uaa or Sp or Poly)_(u)(Xaa)_(x)(±C)(±G)RGDMF(±G)(±C)(Zaa)_(z)[SEQ ID NO:27] (Xaa)_(x)(±C)(±G)RGDMF(±G)(±C)(Zaa)_(z)(Uaa or Sp orPoly)_(u) [SEQ ID NO:27] CS-1 Peptide DELPQLVTLPHPNLHGPEILDVPST [SEQ IDNO:17] (±C)DELPQLVTLPHPNLHGPEILDVPST(±C) [SEQ ID NO:28](Xaa)_(x)DELPQLVTLPHPNLHGPEILDVPST(Zaa)_(z) [SEQ ID NO:28](Xaa)_(x)(±C)DELPQLVTLPHPNLHGPEILDVPST(±C)(Zaa)_(z) [SEQ ID NO:28](±C)(Xaa)_(x)DELPQLVTLPHPNLHGPEILDVPST(Zaa)_(z)(±C) [SEQ ID NO:28] (Uaaor Sp or Poly)_(u)(Xaa)_(x)(±C)DELPQLVTLPHPNLHGPEILDVPST(Zaa)_(z)(±C)[SEQ ID NO:28] (Uaa or Sp orPoly)_(u)(Xaa)_(x)(±C)DELPQLVTLPHPNLHGPEILDVPST(±C)(Zaa)_(z) [SEQ IDNO:28] (±C)(Xaa)_(x)DELPQLVTLPHPNLHGPEILDVPST(±C)(Zaa)_(z)(Uaa or Sp orPoly)_(u) [SEQ ID NO:28](Xaa)_(x)(±C)DELPQLVTLPHPNLHGPEILDVPST(±C)(Zaa)_(z)(Uaa or Sp orPoly)_(u) [SEQ ID NO:28] (Xaa)_(x)DELPQLVTLPHPNLHGPEILDVPST(Zaa)_(z)(Uaaor Sp or Poly)_(u) [SEQ ID NO:28] (Uaa or Sp orPoly)_(u)(Xaa)_(x)DELPQLVTLPHPNLHGPEILDVPST(Zaa)_(z) [SEQ ID NO:28] LDV(active part of CS-1) EILDVPST [SEQ ID NO:29] (±C)EILDVPST(±C) [SEQ IDNO:29] (Xaa)_(x)EILDVPST(Zaa)_(z) [SEQ ID NO:29](Xaa)_(x)(±C)EILDVPST(±C)(Zaa)_(z) [SEQ ID NO:29](±C)(Xaa)_(x)EILDVPST(Zaa)_(z)(±C) [SEQ ID NO:29] (Uaa or Sp orPoly)_(u)(Xaa)_(x)EILDVPST(Zaa)_(z) [SEQ ID NO:29] (Uaa or Sp orPoly)_(u)(Xaa)_(x)(±C)EILDVPST(±C)(Zaa)_(z) [SEQ ID NO:29] (Uaa or Sp orPoly)_(u)(Xaa)_(x)(±C)EILDVPST(Zaa)_(z)(±C) [SEQ ID NO:29](Xaa)_(x)EILDVPST(Zaa)_(z)(Uaa or Sp or Poly)_(u) [SEQ ID NO:29](±C)(Xaa)_(x)EILDVPST(±C)(Zaa)_(z)(Uaa or Sp or Poly)_(u) [SEQ ID NO:29](Xaa)_(x)(±C)EILDVPST(±C)(Zaa)_(z)(Uaa or Sp or Poly)_(u) [SEQ ID NO:29]VSVG 6 mer KFTIVF [SEQ ID NO:30] (±C)KFTIVF(±C) [SEQ ID NO:30](Xaa)_(x)KFTIVF(Zaa)_(z) [SEQ ID NO:30] (Xaa)_(x)(±C)KFTIVF(±C)(Zaa)_(z)[SEQ ID NO:30] (±C)(Xaa)_(x)KFTIVIF(Zaa)_(z)(±C) [SEQ ID NO:30] (Uaa orSp or Poly)_(u)(Xaa)_(x)KFTIVF(Zaa)_(z) [SEQ ID NO:30] (Uaa or Sp orPoly)_(u)(Xaa)_(x)(±C)KFTIVF(±C)(Zaa)_(z) [SEQ ID NO:30] (Uaa or Sp orPoly)_(u)(Xaa)_(x)(±C)KFTIVF(Zaa)_(z)(±C) [SEQ ID NO:30](Xaa)_(x)KFTIVF(Zaa)_(z)(Uaa or Sp or Poly)_(u) [SEQ ID NO:30](±C)(Xaa)_(x)KFTIVF(±C)(Zaa)_(z)(UaaK or Sp or Poly)_(u) [SEQ ID NO:30](Xaa)_(x)(±C)KFTIVF(±C)(Zaa)_(z)(UaaK or Sp or Poly)_(u) [SEQ ID NO:30]CONCATEMERS [(Xaa)_(x)(±C)RGDSP(±C)(Zaa)_(z)]_(p) [SEQ ID NO:26] (Uaa orSp or Poly)_(u)[(Xaa)_(x)(±C)RGDSP(±C)(Zaa)_(z)]_(p) [SEQ ID NO:26][(Xaa)_(x)(±C)RGDMF(±C)(Zaa)_(z)]_(p) [SEQ ID NO:27] (Uaa or Sp orPoly)_(u)[(Xaa)_(m)(±C)RGDMF(±C)(Zaa)_(z)]_(p) [SEQ ID NO:27][(Xaa)_(x)(±C)DELPQLVTLPHPNLHGPEILDVPST(±C)(Zaa)_(z)]_(n) [SEQ ID NO:28](Uaa or Sp orPoly)_(U)[(Xaa)_(x)(±C)DELPQLVTLPHPNLHGPEILDVPST(±C)(Zaa)_(z)]_(p) [SEQID NO:28] [(Xaa)_(x)(±C)DELPQLVTLPHPNLHGPEILDVPST(±C)(Zaa)_(z)]_(p)(Uaaor Sp or Poly)_(u) [SEQ ID NO:28][(Xaa)_(x)(±C)EILDVPST(±C)(Zaa)_(z)]_(p) [SEQ ID NO:29] (Uaa or Sp orPoly)_(uj)(Xaa)_(x)(±C)EILDVPST(±C)(Zaa)_(z)]_(p) [SEQ ID NO:29][(Xaa)_(x)(±C)KFTIVF(±C)(Zaa)_(z)]_(p) [SEQ ID NO:30] (Uaa or Sp orPoly)_(u)[(Xaa)_(x)(±C)KFTIVF(±C)(Zaa)_(z)]_(p) [SEQ ID NO:30](±C)[(Xaa)_(x)RGDSP(Zaa)_(z)(±C) [SEQ ID NO:26] (Uaa or Sp orPoly)_(u)[(Xaa)_(x)(±C)RGDSP(Zaa)_(z)]_(p)(±C) [SEQ ID NO:26](±C)(Xaa)_(x)RGDMF(Zaa)_(z)]_(p)(±C) [SEQ ID NO:27] (Uaa or Sp orPoly)_(u)[(Xaa)_(m)(±C)RGDMF(Zaa)_(z)]_(p)(±C) [SEQ ID NO:27](±C)[(Xaa)_(x)DELPQLVTLPHPNLHGPEILDVPST(Zaa)_(z)]_(p)(±C) [SEQ ID NO:28](Uaa or Sp orPoly)_(U)[(Xaa)_(x)(±C)DELPQLVTLPHPNLHGPEILDVPST(Zaa)_(z)]_(p)(±C) [SEQID NO:28] (±C)[(Xaa)_(x)DELPQLVTLPHPNLHGPEILDVPST(±C)(Zaa)_(z)]_(p)(Uaaor Sp or Poly)_(u) [SEQ ID NO:28] (±C)[(Xaa)_(x)EILDVPST(Zaa)_(p)(±C)[SEQ ID NO:29] (Uaa or Sp orPoly)_(uJ)[(Xaa)_(x)(±C)EILDVPST(Zaa)_(z)]_(p)(±C) [SEQ ID NO:29] (±C)[(Xaa)_(x)KFTIVF(Zaa)_(z)]_(p)(±C) [SEQ ID NO:30] (Uaa or Sp orPoly)_(u)[(Xaa)_(x)(±C)KFTIVF(Zaa)_(z)]_(p)(±C) [SEQ ID NO:30] MIXEDCONCATEMERS [(Baa)_(b)(±C)(±Y orW)(Xaa)_(x)PKKXRKV(Jaa)_(j)RGDMF(±C)(Zaa)_(z)]_(p) [SEQ ID NO:31] (Uaaor Sp or Poly)_(u)[(Baa)_(b)(±C)(Xaa)_(x) (±Y orW)PKKKRKV(Jaa)_(j)RGDMF(±C)(Zaa)_(z)]_(p) [SEQ ID NO:31][(Baa)_(b)(±C)(±Y or W)(Xaa)_(x)PKKKRKV(Jaa)_(j)GRGDSP(±C)(Zaa)_(z)]_(p)[SEQ ID NO:32] (Uaa or Sp or Poly)_(u)[(Baa)_(b)(±C)(±Y orW)(Xaa)_(x)PKKKRKV(Jaa)_(j)RGDSP(±C)(Zaa)_(z)]_(p) [SEQ ID NO:33][(Baa)_(b)(±C)KETIVF(±C)(Xaa)_(x)(±C)(Jaa)_(j)(±Y orW)(Xaa)_(x)PKKKRKV(Zaa)_(z)]_(p) [SEQ ID NO:34] (Uaa or Sp orPoly)[(Baa)_(b)(±C)KFTIVF(±C)(Xaa)_(x)(±C)(Jaa)_(j)(±Y orW)PKKKRKV(Zaa)_(z)]_(p) [SEQ ID NO:34] [(Baa)_(b)(±C)(±Y orW)(Xaa)_(x)GPKKKRKV(Jaa)_(j)EILDVSPT(±C)(Zaa)_(z)]_(p) [SEQ ID NO:35](Uaa or Sp or Poly)_(U)[(Baa)_(b)(±C)(±Y orW)PKKKRKV(Jaa)_(j)EILDVSPT(±C)(Zaa)_(z)]_(p) [SEQ ID NO:36][(Baa)_(b)(±C)KFTIVF(±C)(Xaa)_(x)EILDVPST(±C)(Zaa)_(z)]_(p) [SEQ IDNO:37] (Uaa or Sp orPoly)_(u)[(Baa)_(b)(±C)KETIVF(±C)(Xaa)_(x)EILDVPST(±C)(Zaa)_(z)]_(p)[SEQ ID NO:37] (±C) [(Baa)_(b)(±Y orW)(Xaa)_(x)PKKKRKV(Jaa)_(j)RGDMF(Zaa)_(z)]_(p)(±C) [SEQ ID NO:31] (Uaaor Sp or Poly)_(u)[(Baa)_(b)(±C)(Xaa)_(x)(±Y orW)PKKKRKV(Jaa)_(j)RGDMF(Zaa)_(z)]_(p)(±C) [SEQ ID NO:31] (±C)[(Baa)_(b)(±Y or W)(Xaa)_(x)PKKKRKV(Jaa)_(j)GRGDSP(Zaa)_(z)]_(p)(±C)[SEQ ID NO:32] (Uaa or Sp or Poly)_(u)[(Baa)_(b)(±C)(±Y orW)(Xaa)_(x)PKKKRKV(Jaa)_(j)RGDSP(Zaa)_(z)]_(p)(±C) [SEQ ID NO:33] (±C)[(Baa)_(b)KFTIVF(±C)(Xaa)_(x)(±C)(Jaa)_(j)(±Y orW)(Xaa)_(x)PKKKRKV(Zaa)_(z)]_(p)(±C) [SEQ ID NO:34] (Uaa or Sp orPoly)_(u)[(Baa)_(b)(±C)KFTIVF(±C)(Xaa)_(x)(±C)(Jaa)_(j)(±Y orW)PKKKRKV(Zaa)_(z)]_(p)(±C) [SEQ ID NO:34] (±C) [(Baa)_(b)(±Y orW)(Xaa)_(x)GPKKKRKV(Jaa)_(j)EILDVSPT(Zaa)_(z)]_(p)(±C) [SEQ ID NO:35](Uaa or Sp or Poly)_(U)[(Baa)_(b)(±C)(±Y orW)PKKKRKV(Jaa)_(j)EILDVSPT)(Zaa)_(z)]_(p)(±C) [SEQ ID NO:36] (±C)[(Baa)_(b)KFTIVF(±C)(Xaa)_(x)EILDVPST(Zaa)_(z)]_(p)(±C) [SEQ ID NO:37](Uaa or Sp orPoly)_(u)[(Baa)_(b)(±C)KFTIVF(±C)(Xaa)_(x)EILDVPST)(Zaa)_(z)]_(p)(±C)[SEQ ID NO:37] CIRCULAR LDV (Uaa or Sp orPoly)_(u)(Xaa)_(x)CEILDVPSTC(Zaa)_(z) [SEQ ID NO:38] CIRCULAR RGD,penton base (Uaa or Sp or Poly)_(u)(Xaa)_(x)CHAIRGDTFAC(Zaa)_(z) [SEQ IDNO:39] TAT PEPTIDES CGYGRKKRRQRRRG [SEQ ID NO:40] (±C)CGYGRKKRRQRRRG(±C)[SEQ ID NO:40] (Xaa)_(x)CGYGRKKRRQRRRG(Zaa)_(x) [SEQ ID NO:40](Xaa)_(x)(±C)CGYGRRRRRQRRRG(±C)(Zaa)_(x) [SEQ ID NO:40](±C)(Xaa)_(x)CGYGRKKRRQRRRG(Zaa)_(x)(±C) [SEQ ID NO:40] (Uaa or Sp orPoly)_(u)CGYGRRKRRQRRRG [SEQ ID NO:40 (Uaa or Sp orPoly)_(u)(±C)(Xaa)_(x)(±C)CGYGRKKRRQRRRG(±C)(Zaa)_(x)(±C) [SEQ ID NO:40](±C)(Xaa)_(x)(±C)CGYGRKKRRQRRRG(±C)(Zaa)_(x)(±C)(Uaa or Sp or Poly)_(u)[SEQ ID NO:40] CGYGRKKRRQRRRG(Jaa)_(j)CGYGRKKRRQRRRG [SEQ ID NO:41](CGYGRKKRRQRRRG)_(p) [SEQ ID NO:40] [(Jaa)_(j)CGYGRKKRRQRRRG]_(p) [SEQID NO:40] [(±C)(Xaa)_(x)(±C)CGYGRKKRRQRRRG(±C)(Zaa)_(x)(±C)]_(p) [SEQ IDNO:40] (Uaa or Sp orPoly)_(u)[(±C)(Xaa)_(x)(±C)CGYGRKKRRQRRRG(±C)(Zaa)_(x)(±C)]_(p) [SEQ IDNO:40] [(±C)(Xaa)_(x)(±C)CGYGRKKRRQRRRG(±C)(Zaa)_(x)(±C)]_(p)(Uaa or Spor Poly)_(u) [SEQ ID NO:40] K₁₆CGYGRKKRRQRRRG [SEQ ID NO:42]CGYGRKXRRQRRRGK₁₆ [SEQ ID NO:43] TAT CONCATEMERS NLS-Tat:(±K₁₆)CGYGPKKKRK(Jaa)_(j)CGYGRKKRRQRRRG [SEQ ID NO:44] Tat-NLS:(±K₁₆)CGYGRKKRRQRRRG(Jaa)_(j)CGYGPKKKRK [SEQ ID NO:45] VSVG-Tat:(±K₁₆)KFTTIVFC(Jaa)_(j)CGYGRKKRRQRRRG [SEQ ID NO:46] Tat-VSVG:(±K₁₆)CGYGRKKRRQRRRG(Jaa)_(j)KFTTIVFC [SEQ ID NO:47] Tat-VSVGD6:(±K₁₆)CGYGRKKRRQRRRG(Jaa)_(j)KFTTIVFDDDDDD (±G) [SEQ ID NO:48] Tat-RGD:(±K₁₆)CGYGRKKRRQRRRG(Jaa)_(j)RGDSPC [SEQ ID NO:49](±K₁₆)RGDSPC(Jaa)_(j)CGYGRKKRRQRRRG [SEQ ID NO:50](±K₁₆)CGYGRKKRRQRRRG(Jaa)_(j)GLFEAIAEFIEGGWEGLIEG [SEQ ID NO:51] E5-Tat:(±K₁₆)GLFEAIAEFIEGGWEGLIEG(Jaa)_(j)CGYGRKKRRQRRRG [SEQ ID NO:52]HIS-TEV-Tat: MSYYHHHHHHDYDIPTTENLYFQGS(Jaa)_(j)CGYGRKKRRQRRRG [SEQ IDNO:53] 6HIS-Tat: MSYYHHHHHH(Jaa)_(j)CGYGRKKRRQRRRG [SEQ ID NO:54]

[0163] TABLE 3 Additional Examples of Specific Peptides for TransfectionEnhancement Designation Sequence¹ VSVGD6 GKETIVFDDDDDD(±G) [SEQ IDNO:55] VSVGE5 KFTIVFGGGLFEAIAEFIEGGWEGLIEG [SEQ ID NO:56] E5VSVGGLFEAIAEFIEGGWEGLIEGCKFTIVF [SEQ ID NO:57] NLSE5CGYGGGGGGPKKKRKVGGGLFEAIAEFIEGGWEGLIEG [SEQ ID NO:58] E5NLSGLFEAIAEFIEGGWEGLIEGGGYGGGGGGPKKKRKVGG [SEQ ID NO:59] VSVGNLSKFTTIVFCGYGPKKKRKVGG [SEQ ID NO:60] NLSVSVG CGYGPKKKRKVGGKFTIVF [SEQ IDNO:61] K16VSVGD6 K₁₆GKFTIVFDDDDDD(±G) [SEQ ID NO:62] K16VSVGE5K₁₆KFTIVFGGGLFEAIAEFIEGGWEGLIEG [SEQ ID NO:63] K16E5VSVGK₁₆GLFEAIAEFIEGGWEGLIEGCKFTIVF [SEQ ID NO:64] K16E5NLSK₁₆GLFEAIAEFIEGGWEGLIEGGGYGGGGGGPKKKRKVGG [SEQ ID NO:65] K16VSVGNLSK₁₆KFTTIVFCGYGPKKKRKVGG [SEQ ID NO:66] K16NLSVSVGK₁₆GGCGYGGGPKKKRKVGGKFTIVF [SEQ ID NO:67] K16NLSE5K₁₆GGCGYGGGGGGPKKKRKVGGGLFEAIAEFIEGGWEGLIEG [SEQ ID NO:68] NLSSSDDEATADSQHSTPPKKKRKVGG [SEQ ID NO:69] phosphorylationK₁₆SSDDEATADSQHSTPPKKKRKVGG [SEQ ID NO:70] HIS-TEV-peptidesMSYYHHHHHHDYDIPTTENLYFQGS-peptide [SEQ ID NO:71] HIS-TEV-NLSMSYYHHHHHHDYDIPTTENLYFQGSGYGPKKKRXVGG [SEQ ID NO:72] HIS-TEV-VSVGMSYYHHHHHHDYDIPTTENLYFQGSKFTIVF [SEQ ID NO:73] HIS-TEV-E5MSYYHHHHHHDYDIPTTENLYFQGSGLFEAIAEFIEGGWEGLIEG [SEQ ID NO:74] HIS-TEV-RGDMSYYHHHHHHDYDIPTTENLYFQGSRGDSPC [SEQ ID NO:75 6HIS-peptidesMSYYHHHHHH-peptide [SEQ ID NO:76] 6HIS-NLS MSYYHHHHHHGYGPKKKRKVGG [SEQID NO:77] 6HIS-VSVG MSYYHHHHHHKFTIVF [SEQ ID NO:78] 6HIS-E5MSYYHHHHHHGLFEAIAEFIEGGWEGLIEG [SEQ ID NO:79] 6HIS-RGD MSYYHHHHHHRGDSPC[SEQ ID NO:80] peptide-6HIS peptide-HHHHHH [SEQ ID NO:81]

[0164] TABLE 4 Examples of Spermine-Conjugated Peptides¹Spermine-Conjugated Peptide DesignationSp-5-CO-NH-CH₂-CO-GGGGYGPKKKRKVGG [SEQ ID NO:82] Opf-GG-1Sp-5-CO-NH-CH₂-CO-GYGPKKKRKVG [SEQ ID NO:83] Opf-GG-2Sp-5-CO-NH-CH₂-CO-CGYGPKKKRKVG [SEQ ID NO:84] Opf-GG-2-CYSSp-5-CO-NH-CH₂-CO-GRGDMFGG [SEQ ID NO:85] Sp-RGDSp-5-CO-NH-CH₂-CO-YGPKKKRKVGGGGGRGDMFGG [SEQ ID NO:86] Sp-NLSRGDSp-5-CO-NH-CH₂-CO-GYGPKKKRKVGGGGYGPKKKRKVGG [SEQ ID NO:13] Sp-NLSNLSSp-5-CO-NH-CH((CH₂)₄-NH-5-CO-Sp)-CO- [SEQ ID NO:13] Sp₂-NLSNLSGGYGPKKKRKVGGGGYGPKKKRKVGG

[0165] TABLE 5 Unmodified Peptides Tested for Enhancement of“LIPOFECTAMINE” Transfections in Human Fibroplasts by Inclusion inTransfection Medium Enhancement Peptide Amount Designation Sequence inTransfect. μM E5 GLFEAIAEFIEGGWEGLIEG [SEQ ID NO:88] See FIG. 1 0.1 K5GLFKAIAKFIKGGWKGLIKG [SEQ ID NO:89] ″ 5   HApep GLFGAIAGFIENGWEGMIDG[SEQ ID NO:87] ″ 10  VSVG KFTIVF [SEQ ID NO:30] ″ 1  

[0166] TABLE 6 Comparison of “LIPOFECTAMINE” Transfection +/− Sp-NLSNLSTransfection Conditions: Specific Activity Transfection DNA lipid ngβ-gal/ Protein Yield Reagents (μg) (μl) μg protein μg/Well“LIPOFECTAMINE” 0.4 2 0.07 67.5 “LIPOFECTAMINE” + 0.1 1 0.38 85.5Sp-NLSNLS

[0167] TABLE 7 Enhancement of “LIPOFECTAMINE” Transfections in HumanFibroblasts for Peptides and Derivatized Peptides¹ Peptide Amount (mgDesignation Sequence Enhancement per 0.4 mg DNA) NLS GYGPKKKRKVGG [SEQID NO:90] 7-10 20 Opf-GG-1 Sp-5-CO-GGGGGYGPKKKRKVGG [SEQ ID NO:82] 4  6Opf-GG-2 Sp-5-CO-GGYGPKKKRKVG [SEQ ID NO:83] 5 10 (peak 1) Opf-GG-2partially deblocked² Sp-5-CO-GGYGPKKKRKVG [SEQ ID NO:83 7  2Opf-GG-2-CYS Sp-5-CO-GCGYGPKKKRKVG [SEQ ID NO:84] 5 10 (peak 1)Opf-GG-2-CYS partially deblocked² Sp-5-CO-GCGYGPKKKRKVG [SEQ ID NO:84] 720 SpRGD Sp-5-CO-GGRGDMFGG [SEQ ID NO:85]  5³  12³ SpNLSRGDSp-5-CO-GYGPKKKRKVGGGGGRGDMFGG [SEQ ID NO:86] 4 20 SpNLSNLSSp-5-CO-GGYGPKKKRKVGGGGYGPKKKRKVGG [SEQ ID NO:13] 7-10  4 K16NLSK₁₆GGCGYGPKKKRKVGG [SEQ ID NO:91] 5 10 (peak 1) K16NLS partiallydeblocked² K₁₆GGCGYGPKKKRKVGG [SEQ ID NO:91] 6    0.5 (peak 2) K16NLSRGDK₁₆CGYGPKKKRKVGGGGRGDSP [SEQ ID NO:92] 10   2 K16RGD K₁₆GGRGDSPCG [SEQID NO:93] 10   2 RGDK16 GRGDSPCGGK₁₆ [SEQ ID NO:94] 6  5 K16 K₁₆ [SEQ IDNO:4] 2    0.5 G61934P CGYGPKKKRKVGGGGRGDSPCG [SEQ ID NO:95] 8 20 NLSRGD# blocking group) that remains on the peptide is an Mtr group which is aconventional amino acid blocking group for automated peptide synthesis.

[0168] TABLE 8 Enhancement of DMRIE-C Transfections by Peptides in HumanSuspension Cells (K562 or JurkatCells)¹ Peptide Enhancement- AmountDMRIE-C Peptide/Cell Type Fold μg/0.4 μg DNA μL E5:GLFEAIAIEFIEGGWEGLIEG [SEQ ID NO:113 3 5 1.6 K562 1.2 5 1.6 JurkatK16NLS (peak 2) 1 — 1.6 K562 or Jurkat K16NLSRGD 1 — 1.6 K562 or JurkatK16NLSRGD + E5 1.8 2.5 + 2.5 1.6 K562

[0169] TABLE 9 Enhancement of Dendrimer-Mediated Transfection byPeptides and Spermine-Conjugated Peptides in COS-7 Cells TransfectionAgent Peptide Agent RLU Lipofectamine None 32971 Lipofectamine K16NLS(peak 2) 164105  Lipofectamine Sp-NLSNLS 200447  Lipofectamine NLS224029  G7 (EDA) None  478 G7 (EDA) K16NLS (peak 2)  3423 G7 (EDA)Sp-NLSNLS  2832 G7 (EDA) NLS  2749 G9 (EDA) None  518 G9 (EDA) K16NLS(peak 2)  2297 G9 (EDA) Sp-NLSNLS  1702 G9 (EDA) NLS  1747 Arg DMER None29139 Arg DMER K16NLS (peak 2) 63307 Arg DMER Sp-NLSNLS 56548 Arg DMERNLS 84209 Lys DMER None  2448 Lys DMER K16NLS (peak 2) 20847 Lys DMERSp-NLSNLS 17203 Lys DMER NLS 17689 “COMB BURST” None 18453 “COMB BURST”K16NLS (peak 2) 28562 “COMB BURST” Sp-NLSNLS 23503 “COMB BURST” NLS29639

[0170] TABLE 10 Effect of Sp-NLSNLS on transfection by monocationiclipid reagents. Peak activity (ng b-gal/cm²) Lipid Cell Lipid aloneLipid + SpNLSNLS Lipofectin CHO-K1 70 805 Lipofectin CHO-K1 53 374Lipofectin CHO-K1 20 388 Lipofectin CHO-K1 90 688 Lipofectin HT29 5 56Lipofectin HT29 36 68 Lipofectin HT29 20 31 Lipofectin COS7 4.7 167Lipofectin human fibroblasts 1 97 Lipofectin human fibroblasts 2 197Lipofectin BHK-21 39 497 LipofectACE CHO-K1 0 53 LipofectACE HT29 0 4DMRIE-DOPE human fibroblasts 1.4 9.5

[0171] TABLE 11 Effect of Sp-NLSNLS on transfection by polycationiclipid reagents. Peak activity (ng b-gal/cm²) Lipid + Lipid Cell Lipidalone SpNLSNLS “CELLFECTIN” CHO-K1 21 48 “CELLFECTIN” CHO-K1 190 235“CELLFECTIN” NIH3T3 3.5 14 “CELLFECTIN” human fibroblasts 0.15 2.3 TMDOSCHO-K1 1.4 2.9 TMDOS NIH3T3 0.2 1.3 TMDOS human fibroblasts 0.01 0.1DOSPER human fibroblasts 1.4 5.4 “MULTIFECTOR” human fibroblasts 0.917.6

[0172] TABLE 12 Effect of Sp-NLSNLS on transfection by the dendrimer“SUPERFECT.” Peak activity (ng b-gal/cm²) “SUPERFECT” SUPERFECT + Cellalone SpNLSNLS COS-7 33 84

[0173] TABLE 13 Effect of TAT and spermine-TAT peptides on transfectionby “LIPOFECTAMINE.” ng b-Gal Peak Peak per ug ng b-Gal lipid peptideSample Protein per cm2 (ul) (ug) “LIPOFECTAMINE” alone 0.93 104.47 1.0N/A TAT alone 0.00 0.1 N/A 2.0 sp-TAT alone 0.00 0.06 N/A 0.5 TATw/“LIPOFECTAMINE” 3.73 433.05 1.0 1.0 sp-TAT 3.07 312.77 2.0 0.5w/“LIPOFECTAMINE”

[0174] TABLE 14 Effect of including receptor-ligand proteins withSp-NLSNLS in DNA pre-complexeson transfection by LipofectAMINE andLipofectin. Peak activity (ng β- Lipid: ligand Cell gal/cm²)“LIPOFECTAMINE:” no ligand human fibroblasts 22.9 Insulin humanfibroblasts 24.3 Transferrin human fibroblasts 24.5 Insulin +Transferrin human fibroblasts 31.3 no ligand CHO-K1 358 Insulin CHO-K1452 Transferrin CHO-K1 433 Insulin + Transferrin CHO-K1 460 no ligandhuman fibroblasts 16.8 Insulin human fibroblasts 34.1 no ligand humanfibroblasts 76.7 Insulin human fibroblasts 86.9 “LIPOFECTIN:” no ligandhuman fibroblasts 17.7 Insulin human fibroblasts 22.7 Transferrin humanfibroblasts 30.6 Insulin + Transferrin human fibroblasts 37.0 no ligandCHO-K1 29 Insulin CHO-K1 31 Transferrin CHO-K1 44 Insulin + TransferrinCHO-K1 35 DMRIE-DOPE: no ligand human fibroblasts 9.5 Insulin humanfibroblasts 20.7

[0175] TABLE 15 Effect of an adhesion Protein Fragment on transfectionby LipofectAMINE Reagent. Peak activity (ng β-gal/cm) LipofectAMINE +Cell LipofectAMINE alone Retronectin COS-7 39.5 138.4

[0176]

1 120 10 amino acids amino acid not relevant linear peptide NO NO 1 HisAla Ile Arg Gly Asp Thr Phe Ala Thr 1 5 10 7 amino acids amino acid notrelevant linear peptide NO NO 2 Pro Lys Lys Lys Arg Lys Val 1 5 8 aminoacids amino acid not relevant linear peptide NO NO 3 Pro Lys Lys Lys LysArg Lys Val 1 5 20 amino acids amino acid not relevant linear peptide NONO Modified-site 2..20 /product= “OTHER” /note= “ANY OR ALL OF THE AMINOACIDS 2-20 CAN BE PRESENT OR ABSENT” 4 Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Lys Lys Lys Lys 20 20 aminoacids amino acid not relevant linear peptide Modified-site 2..20/product= “OTHER” /note= “ANY OR ALL OF THE AMINO ACIDS 2-20 CAN BEPRESENT OR ABSENT” 5 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg ArgArg Arg Arg 1 5 10 15 Arg Arg Arg Arg 20 40 amino acids amino acid notrelevant linear peptide Modified-site 3..40 /product= “OTHER” /note=“ANY Lsy-Arg PAIR FROM AMINO ACID 3-40 CAN BE PRESENT OR ABSENT” 6 LysArg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg 1 5 10 15Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg Lys Arg 20 25 30Lys Arg Lys Arg Lys Arg Lys Arg 35 40 20 amino acids amino acid notrelevant linear peptide Modified-site 2..20 /product= “OTHER” /note=“ANY OR ALL OF THE AMINO ACIDS 2-20 CAN BE PRESENT OR ABSENT” 7 Gly GlyGly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 GlyGly Gly Gly 20 7 amino acids amino acid not relevant linear peptide NONO 8 Gly Arg Gly Asp Ser Pro Cys 1 5 23 amino acids amino acid notrelevant linear peptide Modified-site 1..20 /product= “OTHER” /note=“ANY OR ALL OF THE AMINO ACIDS 1-20 CAN BE PRESENT OR ABSENT” 9 Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 LysLys Lys Lys Arg Gly Asp 20 19 amino acids amino acid not relevant linearpeptide 10 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys 1 5 10 15 Arg Gly Asp 7 amino acids amino acid not relevant linearpeptide NO NO 11 Lys Phe Thr Ile Val Phe Cys 1 5 12 amino acids aminoacid not relevant linear peptide NO NO 12 Cys Gly Trp Gly Pro Lys LysLys Arg Lys Val Gly 1 5 10 26 amino acids amino acid not relevant linearpeptide NO NO 13 Gly Gly Tyr Gly Pro Lys Lys Lys Arg Lys Val Gly Gly GlyGly Tyr 1 5 10 15 Gly Pro Lys Lys Lys Arg Lys Val Gly Gly 20 25 6 aminoacids amino acid not relevant linear peptide NO NO 14 Arg Gly Asp SerPro Cys 1 5 4 amino acids amino acid not relevant linear peptide NO NO15 Arg Glu Asp Val 1 4 amino acids amino acid not relevant linearpeptide NO NO 16 Arg Gly Asp Val 1 25 amino acids amino acid notrelevant linear peptide NO NO 17 Asp Glu Leu Pro Gln Leu Val Thr Leu ProHis Pro Asn Leu His Gly 1 5 10 15 Pro Glu Ile Leu Asp Val Pro Ser Thr 2025 4 amino acids amino acid not relevant linear peptide NO NO 18 Arg GlyAsp Asn 1 4 amino acids amino acid not relevant linear peptide NO NO 19Arg Gly Asp Met 1 4 amino acids amino acid not relevant linear peptideNO NO 20 Arg Gly Asp Thr 1 4 amino acids amino acid not relevant linearpeptide NO NO Modified-site 4 /product= “OTHER” /note= “Xaa AT POSITION4 CAN BE ANY AMINO ACID” 21 Arg Gly Asp Xaa 1 4 amino acids amino acidnot relevant linear peptide NO NO 22 Arg Gly Asp Ser 1 4 amino acidsamino acid not relevant linear peptide NO NO 23 Arg Gly Asp Ala 1 33amino acids amino acid not relevant linear peptide Modified-site 1/product= “OTHER” /note= “C AT POSITION 1 CAN BE ABSENT” Modified-site 3/product= “OTHER” /note= “Xaa AT POSITION 3 CAN BE TYR OR TRP OR CAN BEABSENT” Modified-site 14..33 /product= “OTHER” /note= “ANY OR ALL OF THEGLY AT POSITIONS 14 TO 33 CAN BE ABSENT” 24 Cys Gly Xaa Gly Pro Lys LysLys Arg Lys Val Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly Gly GlyGly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30 Gly 48 amino acidsamino acid not relevant linear peptide Modified-site 1..20 /product=“OTHER” /note= “Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACIDS AND ANY ALLOF THESE AMINO ACIDS CAN BE ABSENT ” Modified-site 28 /product= “OTHER”/note= “Xaa AT POSITION 28 CAN BE TYR OR TRP OR CAN BE ABSENT”Modified-site 29..48 /product= “OTHER” /note= “Xaa AT POSITIONS 29-48CAN BE ANY AMINO ACIDS OR CAN ABSENT” 25 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Pro Lys LysLys Arg Lys Val Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 90 amino acids amino acidnot relevant linear peptide Modified-site 1..20 /product= “OTHER” /note=“Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACIDS OR ANY OF THESE AMINOACIDS CAN BE ABSENT” Modified-site 21 /product= “OTHER” /note= “CYS ATPOSITON 21 CAN BE ABSENT” Modified-site 22 /product= “OTHER” /note= “XaaAT POSITION 22 CAN BE TYR OR TRP OR CAN BE ABSENT” Modified-site 23..42/product= “OTHER” /note= “Xaa AT POSITIONS 23-42 CAN BE ANY AMINO ACIDSOR ANY OF THESE AMINO ACIDS CAN BE ABSENT” Modified-site 50 /product=“OTHER” /note= “CYS AT POSITION 50 CAN BE ABSENT” Modified-site 51..70/product= “OTHER” /note= “Xaa AT POSITIONS 51 TO 79 CAN BE ANY AMINOACIDS OR ANY OF THESE AMINO ACIDS CAN BE ABSENT” Modified-site 71..90/product= “OTHER” /note= “Xaa AT POSITIONS 71-90 CAN BE ANY CATIONICAMINO ACIDS OR ANY OF THESE AMINO ACIDS CAN BE ABSENT” 26 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa XaaXaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Lys Lys Lys Arg Lys 35 40 45 Val CysXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 70 amino acids amino acid notrelevant linear peptide Modified-site 1 /product= “OTHER” /note= “CYS ATPOSITION 1 CAN BE ABSENT” Modified-site 2..21 /product= “OTHER” /note=“Xaa AT POSITIONS 2-21 CAN BE ANY AMINO ACIDS OR ANY OF THESE AMNIONACIDS CAN BE ABSENT” Modified-site 22 /product= “OTHER” /note= “Xaa ATPOSITION 22 CAN BE TYR OR TRP OR CAN BE ABSENT” Modified-site 23..42/product= “OTHER” /note= “Xaa AT POSITIONS 23 TO 42 CAN BE ANY AMNIOACIDS OR ANY OF THESE AMINO ACIDS CAN BE ABSENT” Modified-site 50..69/product= “OTHER” /note= “Xaa AT POSITIONS 50 TO 69 CAN BE ANY AMINOACIDS OR ANY OF THESE AMINO ACIDS CAN BE ABSENT” Modified-site 70/product= “OTHER” /note= “CYS AT POSITION 70 CAN BE ABSENT” 27 Cys XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Lys Lys Lys Arg Lys 35 40 45 ValXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 XaaXaa Xaa Xaa Xaa Cys 65 70 90 amino acids amino acid not relevant linearpeptide Modified-site 1 /product= “OTHER” /note= “CYS AT POSITION 1 CANBE ABSENT” Modified-site 2..21 /product= “OTHER” /note= “Xaa ATPOSITIONS 2-21 CAN BE ANY AMINO ACIDS OR ANY OF THESE AMINO ACIDS CAN BEABSENT” Modified-site 22 /product= “OTHER” /note= “Xaa AT POSITION 22CAN BE TYR OR TRP OR CAN BE ABSENT” Modified-site 23..42 /product=“OTHER” /note= “Xaa AT POSITIONS 23-42 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 50 /product= “OTHER” /note= “CYS AT POSITION 50CAN BE ABSENT” Modified-site 51..70 /product= “OTHER” /note= “Xaa ATPOSITIONS 51 TO 70 CAN BE ANY AMINO ACIDS AND CAN BE ABSENT”Modified-site 71..90 /product= “OTHER” /note= “Xaa AT POSITIONS 71-90CAN BE ANY CATIONIC AMINO ACIDS OR CAN BE ABSENT” 28 Cys Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Pro Lys Lys Lys Arg Lys 35 40 45 Val Cys Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 90 amino acids amino acid not relevantlinear peptide Modified-site 1..20 /product= “OTHER” /note= “Xaa ATPOSITIONS 1-20 CAN BE ANY CATIONIC AMINO ACIDS OR CAN BE AB...”Modified-site 21..40 /product= “OTHER” /note= “Xaa AT POSITIONS 21 TO 40CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 41 /product=“OTHER” /note= “CYS AT POSITION 41 CAN BE ABSENT” Modified-site 42/product= “OTHER” /note= “Xaa AT POSITION 42 CAN BE TYR OT TRP OR CAN BEABSENT” Modified-site 43..62 /product= “OTHER” /note= “Xaa AT POSITIONS43 TO 62 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 70..89/product= “OTHER” /note= “Xaa AT POSITIONS 70 TO 89 CAN BE ANY AMINOACIDS OR CAN BE ABSENT” Modified-site 90 /product= “OTHER” /note= “CYSAT POSITION 90 CAN BE ABSENT” 29 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa CysXaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Pro Lys 50 55 60 Lys Lys Arg Lys Val Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Cys 85 90 90 amino acids amino acid not relevant linear peptideModified-site 1..20 /product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CANBE ANY CATIONIC AMINO ACID OR CAN BE ABSENT” Modified-site 21..40/product= “OTHER” /note= “Xaa AT POSITIONS 21 TO 40 CAN BE ANY AMINOACIDS OR CAN BE ABSENT” Modified-site 41 /product= “OTHER” /note= “CYSAT POSITION 41 CAN BE ABSENT” Modified-site 42 /product= “OTHER” /note=“Xaa AT POSITION 42 CAN BE TYR OR TRP OR CAN BE ABSENT” Modified-site43..62 /product= “OTHER” /note= “Xaa AT POSITIONS 43 TO 62 CAN BE ANYAMINO ACIDS OR CAN BE ABSENT” Modified-site 70 /product= “OTHER” /note=“CYS AT POSITION 70 CAN BE ABSENT” Modified-site 71..90 /product=“OTHER” /note= “Xaa AT POSITIONS 71 TO 90 CAN BE ANY AMINO ACIDS OR CANBE ABSENT” 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa XaaXaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Pro Lys 50 55 60 Lys Lys Arg Lys Val Cys Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 5amino acids amino acid not relevant linear peptide 31 Arg Gly Asp SerPro 1 5 69 amino acids amino acid not relevant linear peptideModified-site 1..20 /product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CANBE ANY CATIONIC AMINO ACIDS OR CAN BE AB...” Modified-site 21..40/product= “OTHER” /note= “Xaa AT POSITIONS 21-40 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” Modified-site 41 /product= “OTHER” /note= “CYS ATPOSITION 41 CAN BE ABSENT” Modified-site 42 /product= “OTHER” /note=“GLY AT POSITION 42 CAN BE ABSENT” Modified-site 48 /product= “OTHER”/note= “GLY AT POSITION 48 CAN BE ABSENT” Modified-site 49 /product=“OTHER” /note= “CYS AT POSITION 49 CAN BE ABSENT” Modified-site 50..69/product= “OTHER” /note= “Xaa AT POSITIONS 50-69 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” 32 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Gly Arg GlyAsp Ser Pro Gly 35 40 45 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa 65 69 amino acids aminoacid not relevant linear peptide Modified-site 1..20 /product= “OTHER”/note= “Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 21 /product= “OTHER” /note= “CYS AT POSITION 21 CAN BEABSENT” Modified-site 22 /product= “OTHER” /note= “GLY AT POSITION 22CAN BE ABSENT” Modified-site 28 /product= “OTHER” /note= “GLY ATPOSITION 28 CAN BE ABSENT” Modified-site 29 /product= “OTHER” /note=“CYS AT POSITION 29 CAN BE ABSENT” Modified-site 30..49 /product=“OTHER” /note= “Xaa AT POSITIONS 30-49 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 50..69 /product= “OTHER” /note= “Xaa AT POSITIONS50 -69 CAN BE ANY CATIONIC AMINO ACIDS” 33 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys GlyArg Gly Asp Ser Pro Gly Cys Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa 6569 amino acids amino acid not relevant linear peptide Modified-site1..20 /product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CAN BE ANYCATIONIC AMINO ACIDS OR CAN BE AB...” Modified-site 21..40 /product=“OTHER” /note= “Xaa AT POSITIONS 21-40 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 41 /product= “OTHER” /note= “CYS AT POSITION 41CAN BE ABSENT” Modified-site 42 /product= “OTHER” /note= “GLY ATPOSITION 42 CAN BE ABSENT” Modified-site 48 /product= “OTHER” /note=“GLY AT POSITION 48 CAN BE ABSENT” Modified-site 49..68 /product=“OTHER” /note= “Xaa AT POSITIONS 49-68 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 69 /product= “OTHER” /note= “CYS AT POSITION 69CAN BE ABSENT” 34 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Gly Arg GlyAsp Ser Pro Gly 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Cys 65 69 amino acids aminoacid not relevant linear peptide Modified-site 1 /product= “OTHER”/note= “CYS AT POSITION 1 CAN BE ABSENT” Modified-site 2..21 /product=“OTHER” /note= “Xaa AT POSITIONS 2-21 CAN BE ANY AMINO ACID OR CAN BEABSENT” Modified-site 22 /product= “OTHER” /note= “GLY AT POSITION 22CAN BE ABSENT” Modified-site 28 /product= “OTHER” /note= “GLY ATPOSITION 28 CAN BE ABSENT” Modified-site 29 /product= “OTHER” /note=“CYS AT POSITION 29 CAN BE ABSENT” Modified-site 30..49 /product=“OTHER” /note= “Xaa AT POSITIONS 30-49 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 50..69 /product= “OTHER” /note= “Xaa AT POSITIONS50-69 CAN BE ANY CATIONIC AMINO ACIDS OR CAN BE ABSENT” 35 Cys Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa XaaXaa Xaa Xaa Gly Arg Gly Asp Ser Pro Gly Cys Xaa Xaa Xaa 20 25 30 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa XaaXaa Xaa Xaa 65 5 amino acids amino acid not relevant linear peptide 36Arg Gly Asp Met Phe 1 5 49 amino acids amino acid not relevant linearpeptide Modified-site 1..20 /product= “OTHER” /note= “Xaa AT POSITIONS1-20 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 21 /product=“OTHER” /note= “CYS AT POSITION 21 CAN BE ABSENT” Modified-site 22/product= “OTHER” /note= “GLY AT POSITION 22 CAN BE ABSENT”Modified-site 28 /product= “OTHER” /note= “GLY AT POSITION 28 CAN BEABSENT” Modified-site 29 /product= “OTHER” /note= “CYS AT POSITION 29CAN BE ABSENT” Modified-site 30..49 /product= “OTHER” /note= “Xaa ATPOSITIONS 30-49 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” 37 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa XaaXaa Xaa Cys Gly Arg Gly Asp Met Phe Gly Cys Xaa Xaa Xaa 20 25 30 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa 47amino acids amino acid not relevant linear peptide Modified-site 1/product= “OTHER” /note= “CYS AT POSITION 1 CAN BE ABSENT” Modified-site2..21 /product= “OTHER” /note= “Xaa AT POSITIONS 2-21 CAN BE ANY AMINOACIDS OR CAN BE ABSENT” Modified-site 27..46 /product= “OTHER” /note=“Xaa AT POSITIONS 27-46 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 47 /product= “OTHER” /note= “CYS AT POSITION 47 CAN BEABSENT” 38 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Arg Gly Asp Met Phe Xaa Xaa Xaa XaaXaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaCys 35 40 45 69 amino acids amino acid not relevant linear peptideModified-site 1..20 /product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CANBE ANY CATIONIC AMINO ACIDS OR CAN BE AB...” Modified-site 21..40/product= “OTHER” /note= “Xaa AT POSITIONS 21-40 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” Modified-site 41 /product= “OTHER” /note= “CYS ATPOSITION 41 CAN BE ABSENT” Modified-site 42 /product= “OTHER” /note=“GLY AT POSITION 42 CAN BE ABSENT” Modified-site 48 /product= “OTHER”/note= “GLY AT POSITION 48 CAN BE ABSENT” Modified-site 49..68 /product=“OTHER” /note= “Xaa AT POSITIONS 49-68 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 69 /product= “OTHER” /note= “CYS AT POSITION 69CAN BE ABSENT” 39 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Gly Arg GlyAsp Met Phe Gly 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Cys 65 69 amino acids aminoacid not relevant linear peptide Modified-site 1 /product= “OTHER”/note= “CYS AT POSITION 1 CAN BE ABSENT” Modified-site 2..21 /product=“OTHER” /note= “Xaa AT POSITIONS 2-21 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 22 /product= “OTHER” /note= “GLY AT POSITION 22CAN BE ABSENT” Modified-site 28 /product= “OTHER” /note= “GLY ATPOSITION 28 CAN BE ABSENT” Modified-site 29 /product= “OTHER” /note=“CYS AT POSITION 29 CAN BE ABSENT” Modified-site 30..49 /product=“OTHER” /note= “Xaa AT POSITIONS 30-49 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 50..69 /product= “OTHER” /note= “Xaa AT POSITIONS50-69 CAN BE ANY CATIONIC AMINO ACIDS OR CAN BE ABSENT” 40 Cys Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa XaaXaa Xaa Xaa Gly Arg Gly Asp Met Phe Gly Cys Xaa Xaa Xaa 20 25 30 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa XaaXaa Xaa Xaa 65 69 amino acids amino acid not relevant linear peptideModified-site 1..20 /product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CANBE ANY CATIONIC AMINO ACIDS OR CAN BE AB...” Modified-site 21..40/product= “OTHER” /note= “Xaa AT POSITIONS 21-40 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” Modified-site 41 /product= “OTHER” /note= “CYS ATPOSITION 41 CAN BE ABSENT” Modified-site 42 /product= “OTHER” /note=“GLY AT POSITION 42 CAN BE ABSENT” Modified-site 48 /product= “OTHRE”/note= “GLY AT POSITION 48 CAN BE ABSENT” Modified-site 49 /product=“OTHER” /note= “CYS AT POSITION 49 CAN BE ABSENT” Modified-site 50..69/product= “OTHER” /note= “Xaa AT POSITIONS 50-69 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” 41 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Gly Arg GlyAsp Met Phe Gly 35 40 45 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa 65 69 amino acids aminoacid not relevant linear peptide Modified-site 1..20 /product= “OTHER”/note= “Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 21 /product= “OTHER” /note= “CYS AT POSITION 21 CAN BEABSENT” Modified-site 22 /product= “OTHER” /note= “GLY AT POSITION 22CAN BE ABSENT” Modified-site 28 /product= “OTHER” /note= “GLY ATPOSITION 28 CAN BE ABSENT” Modified-site 29 /product= “OTHER” /note=“CYS AT POSITION 29 CAN BE ABSENT” Modified-site 30..49 /product=“OTHER” /note= “Xaa AT POSITIONS 30-49 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 50..69 /product= “OTHER” /note= “Xaa AT POSITIONS50-69 CAN BE ANY CATIONIC AMINO ACIDS OR CAN BE ABSENT” 42 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa XaaXaa Xaa Cys Gly Arg Gly Asp Met Phe Gly Cys Xaa Xaa Xaa 20 25 30 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa XaaXaa Xaa Xaa 65 67 amino acids amino acid not relevant linear peptideModified-site 1 /product= “OTHER” /note= “CYS AT POSITION 1 CAN BEABSENT” Modified-site 2..21 /product= “OTHER” /note= “Xaa AT POSITIONS2-21 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 47..66/product= “OTHER” /note= “Xaa AT POSITIONS 47-66 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” Modified-site 67 /product= “OTHER” /note= “CYS ATPOSITION 67 CAN BE ABSENT” 43 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Asp Glu Leu ProGln Leu Val Thr Leu Pro His 20 25 30 Pro Asn Leu His Gly Pro Glu Ile LeuAsp Val Pro Ser Thr Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Cys 65 87 amino acids aminoacid not relevant linear peptide Modified-site 1..20 /product= “OTHER”/note= “Xaa AT POSITIONS 1-20 CAN BE ANY CATIONIC AMINO ACID OR CAN BEABSENT” Modified-site 21..40 /product= “OTHER” /note= “Xaa AT POSITIONS21-40 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 41/product= “OTHER” /note= “CYS AT POSITION 41 CAN BE ABSENT”Modified-site 67..86 /product= “OTHER” /note= “Xaa AT POSITIONS 67-86CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 87 /product=“OTHER” /note= “CYS AT POSITION 87 CAN BE ABSENT” 44 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Cys Asp Glu Leu Pro Gln Leu Val 35 40 45 Thr Leu Pro HisPro Asn Leu His Gly Pro Glu Ile Leu Asp Val Pro 50 55 60 Ser Thr Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa XaaXaa Xaa Xaa Cys 85 87 amino acids amino acid not relevant linear peptideModified-site 1..20 /product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CANBE ANY CATIONIC AMINO ACIDS OR CAN BE AB...” Modified-site 21..40/product= “OTHER” /note= “Xaa AT POSITIONS 21-40 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” Modified-site 41 /product= “OTHER” /note= “CYS ATPOSTION 41 CAN BE ABSENT” Modified-site 67 /product= “OTHER” /note= “CYSAT POSITION 67 CAN BE ABSENT” Modified-site 68..87 /product= “OTHER”/note= “Xaa AT POSITIONS 68-87 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 510 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 2025 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Asp Glu Leu Pro Gln Leu Val 3540 45 Thr Leu Pro His Pro Asn Leu His Gly Pro Glu Ile Leu Asp Val Pro 5055 60 Ser Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 6570 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 87 amino acids amino acid notrelevant linear peptide Modified-site 1 /product= “OTHER” /note= “CYS ATPOSITION 1 CAN BE ABSENT” Modified-site 2..21 /product= “OTHER” /note=“Xaa AT POSITIONS 2-21 CAN BE ANY AMINO ACID OR CAN BE ABSENT”Modified-site 47 /product= “OTHER” /note= “CYS AT POSITION 47 CAN BEABSENT” Modified-site 48..67 /product= “OTHER” /note= “Xaa AT POSITIONS48-67 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 68..87/product= “OTHER” /note= “Xaa AT POSITIONS 68-87 CAN BE ANY CATIONICAMINO ACIDS OR CAN BE ABSENT” 46 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Asp Glu Leu ProGln Leu Val Thr Leu Pro His 20 25 30 Pro Asn Leu His Gly Pro Glu Ile LeuAsp Val Pro Ser Thr Cys Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 8587 amino acids amino acid not relevant linear peptide Modified-site1..20 /product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CAN BE ANY AMINOACIDS OR CAN BE ABSENT” Modified-site 21 /product= “OTHER” /note= “CYSAT POSITION 21 CAN BE ABSENT” Modified-site 47 /product= “OTHER” /note=“CYS AT POSITION 47 CAN BE ABSENT” Modified-site 48..67 /product=“OTHER” /note= “Xaa AT POSITIONS 48-67 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 68..87 /product= “OTHER” /note= “Xaa AT POSITIONS68-87 CAN BE ANY CATIONIC AMNIO ACIDS OR CAN BE ABSENT” 47 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa XaaXaa Xaa Cys Asp Glu Leu Pro Gln Leu Val Thr Leu Pro His 20 25 30 Pro AsnLeu His Gly Pro Glu Ile Leu Asp Val Pro Ser Thr Cys Xaa 35 40 45 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 XaaXaa Xaa Xaa Xaa Xaa Xaa 85 8 amino acids amino acid not relevant linearpeptide 48 Glu Ile Leu Asp Val Pro Ser Thr 1 5 50 amino acids amino acidnot relevant linear peptide Modified-site 1 /product= “OTHER” /note=“CYS AT POSITION 1 CAN BE ABSENT” Modified-site 21 /product= “OTHER”/note= “Xaa AT POSITIONS 2-21 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 30..49 /product= “OTHER” /note= “Xaa AT POSITIONS 30-49CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 50 /product=“OTHER” /note= “CYS AT POSITION 50 CAN BE ABSENT” 49 Cys Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa XaaXaa Glu Ile Leu Asp Val Pro Ser Thr Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Cys 50 70amino acids amino acid not relevant linear peptide Modified-site 1..20/product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CAN BE ANY CATIONICAMINO ACIDS OR CAN BE AB...” Modified-site 21..40 /product= “OTHER”/note= “Xaa AT POSITIONS 21-40 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 41 /product= “OTHER” /note= “CYS AT POSITION 41 CAN BEABSENT” Modified-site 50 /product= “OTHER” /note= “CYS AT POSITION 50CAN BE ABSENT” Modified-site 51..70 /product= “OTHER” /note= “Xaa ATPOSITIONS 51-70 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” 50 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Cys Glu Ile Leu Asp Val Pro Ser 35 40 45 Thr CysXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa XaaXaa Xaa Xaa Xaa 65 70 70 amino acids amino acid not relevant linearpeptide Modified-site 1..20 /product= “OTHER” /note= “Xaa AT POSITIONS1-20 CAN BE ANY CATIONIC AMINO ACIDS OR CAN BE AB...” Modified-site21..40 /product= “OTHER” /note= “Xaa AT POSITIONS 21-40 CAN BE ANY AMINOACIDS OR CAN BE ABSENT” Modified-site 41 /product= “OTHER” /note= “CYSAT POSITION 41 CAN BE ABSENT” Modified-site 50..69 /product= “OTHER”/note= “Xaa AT POSITIONS 50-69 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 70 /product= “OTHER” /note= “CYS AT POSITION 70 CAN BEABSENT” 51 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Glu Ile Leu Asp ValPro Ser 35 40 45 Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Cys 65 70 70 amino acids amino acidnot relevant linear peptide Modified-site 1 /product= “OTHER” /note=“CYS AT POSITION 1 CAN BE ABSENT” Modified-site 2..21 /product= “OTHER”/note= “Xaa AT POSITIONS 2-21 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 30 /product= “OTHER” /note= “CYS AT POSITION 30 CAN BEABSENT” Modified-site 31..50 /product= “OTHER” /note= “Xaa AT POSITIONS31-50 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 51..70/product= “OTHER” /note= “Xaa AT POSITIONS 51-70 AN BE ANY CATIONICAMINO ACIDS OR CAN BE AB...” 52 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Glu Ile Leu AspVal Pro Ser Thr Cys Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa 65 70 70amino acids amino acid not relevant linear peptide Modified-site 1..20/product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” Modified-site 21 /product= “OTHER” /note= “CYS ATPOSITION 21 CAN BE ABSENT” Modified-site 30 /product= “OTHER” /note=“CYS AT POSITION 50 CAN BE ABSENT” Modified-site 31..50 /product=“OTHER” /note= “Xaa AT POSITIONS 31-50 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 51..70 /product= “OTHER” /note= “Xaa AT POSITIONS51-70 CAN BE ANY CATIONIC AMINO ACIDS OR CAN BE ABSENT” 53 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa XaaXaa Xaa Cys Glu Ile Leu Asp Val Pro Ser Thr Cys Xaa Xaa 20 25 30 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa XaaXaa Xaa Xaa Xaa 65 70 6 amino acids amino acid not relevant linearpeptide 54 Lys Phe Thr Ile Val Phe 1 5 48 amino acids amino acid notrelevant linear peptide Modified-site 1 /product= “OTHER” /note= “CYS ATPOSITION 1 CAN BE ABSENT” Modified-site 2..21 /product= “OTHER” /note=“Xaa AT POSITIONS 2-21 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 28..47 /product= “OTHER” /note= “Xaa AT POSITIONS 28-47CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 48 /product=“OTHER” /note= “CYS AT POSITION 48 CAN BE ABSENT” 55 Cys Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa XaaXaa Lys Phe Thr Ile Val Phe Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 45 68 amino acidsamino acid not relevant linear peptide Modified-site 1..20 /product=“OTHER” /note= “Xaa AT POSITIONS 1-20 CAN BE ANY CATIONIC AMINO ACIDS ORCAN BE AB...” Modified-site 21..40 /product= “OTHER” /note= “Xaa ATPOSITIONS 21-40 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site41 /product= “OTHER” /note= “CYS AT POSITION 41 CAN BE ABSENT”Modified-site 48 /product= “OTHER” /note= “CYS AT POSITION 48 CAN BEABSENT” Modified-site 49..68 /product= “OTHER” /note= “Xaa AT POSITIONS49-68 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” 56 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Cys Lys Phe Thr Ile Val Phe Cys 35 40 45 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa65 68 amino acids amino acid not relevant linear peptide Modified-site1..20 /product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CAN BE ANYCATIONIC AMINO ACID OR CAN BE ABSENT” Modified-site 21..40 /product=“OTHER” /note= “Xaa AT POSITIONS 21-40 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 41 /product= “OTHER” /note= “CYS AT POSITION 41CAN BE ABSENT” Modified-site 48..67 /product= “OTHER” /note= “Xaa ATPOSITIONS 48-67 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site68 /product= “OTHER” /note= “CYS AT POSITION 68 CAN BE ABSENT” 57 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Lys Phe Thr Ile Val Phe Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Cys 65 68 amino acids amino acid not relevant linear peptideModified-site 1 /product= “OTHER” /note= “CYS AT POSITION 1 CAN BEABSENT” Modified-site 2..21 /product= “OTHER” /note= “Xaa AT POSITIONS2-21 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 28 /product=“OTHER” /note= “CYS AT POSITION 28 CAN BE ABSENT” Modified-site 29..48/product= “OTHER” /note= “Xaa AT POSITIONS 29-48 CAN BE ANY AMNINO ACIDSOR CAN BE ABSENT” Modified-site 49..68 /product= “OTHER” /note= “Xaa ATPOSITIONS 49-68 CAN BE ANY CATIONIC AMINO ACIDS OR CAN BE ABSENT” 58 CysXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15Xaa Xaa Xaa Xaa Xaa Lys Phe Thr Ile Val Phe Cys Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa 65 68 amino acids amino acid not relevant linear peptideModified-site 1..20 /product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CANBE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 21 /product= “OTHER”/note= “CYS AT POSITION 21 CAN BE ABSENT” Modified-site 28 /product=“OTHER” /note= “CYS AT POSITION 28 CAN BE ABSENT” Modified-site 29..48/product= “OTHER” /note= “Xaa AT POSITIONS 29-48 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” Modified-site 49..68 /product= “OTHER” /note= “Xaa ATPOSITIONS 49-68 CAN BE ANY CATIONIC AMINO ACIDS OR CAN BE ABSENT” 59 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15Xaa Xaa Xaa Xaa Cys Lys Phe Thr Ile Val Phe Cys Xaa Xaa Xaa Xaa 20 25 30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60Xaa Xaa Xaa Xaa 65 95 amino acids amino acid not relevant linear peptideModified-site 1..20 /product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CANBE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 21 /product= “OTHER”/note= “CYS AT POSITION 21 CAN BE ABSENT” Modified-site 22 /product=“OTHER” /note= “Xaa AT POSITION 22 CAN BE TYR OR TRP OR CAN BE ABSENT”Modified-site 23..42 /product= “OTHER” /note= “Xaa AT POSITIONS 23-42ANC BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 50..69 /product=“OTHER” /note= “Xaa AT POSITIONS 50-69 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 75 /product= “OTHER” /note= “CYS AT POSITION 75CAN BE ABSENT” Modified-site 76..95 /product= “OTHER” /note= “Xaa ATPOSITIONS 76-95 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” 60 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa XaaXaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Lys Lys Lys Arg Lys 35 40 45 Val XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa XaaXaa Xaa Xaa Arg Gly Asp Met Phe Cys Xaa Xaa Xaa Xaa Xaa 65 70 75 80 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 95amino acids amino acid not relevant linear peptide Modified-site 1..20/product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” Modified-site 21 /product= “OTHER” /note= “CYS ATPOSITION 21 CAN BE ABSENT” Modified-site 22..41 /product= “OTHER” /note=“Xaa AT POSITIONS 22-41 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 42 /product= “OTHER” /note= “Xaa AT POSITION 42 CAN BE TYROR TRP OR CAN BE ABSENT” Modified-site 50..69 /product= “OTHER” /note=“Xaa AT POSITIONS 50-69 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 75 /product= “OTHER” /note= “CYS AT POSITION 75 CAN BEABSENT” Modified-site 76..95 /product= “OTHER” /note= “Xaa AT POSITIONS76-95 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” 61 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa XaaCys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Pro Lys Lys Lys Arg Lys 35 40 45 Val Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa XaaXaa Arg Gly Asp Met Phe Cys Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 96 amino acidsamino acid not relevant linear peptide Modified-site 1..20 /product=“OTHER” /note= “Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 21 /product= “OTHER” /note= “CYS AT POSITION 21CAN BE ABSENT” Modified-site 22 /product= “OTHER” /note= “Xaa ATPOSITION 22 CAN BE TYR OR TRP OR CAN BE ABSENT” Modified-site 23..42/product= “OTHER” /note= “Xaa AT POSITIONS 23-42 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” Modified-site 50..69 /product= “OTHER” /note= “Xaa ATPOSITIONS 50-69 CAN BE ANY AMINO ACIDS OF CAN BE ABSENT” Modified-site76 /product= “OTHER” /note= “CYS AT POSITION 76 CAN BE ABSENT”Modified-site 77..96 /product= “OTHER” /note= “Xaa AT POSITIONS 77-96CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” 62 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Pro Lys Lys Lys Arg Lys 35 40 45 Val Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa GlyArg Gly Asp Ser Pro Cys Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 95 amino acidsamino acid not relevant linear peptide Modified-site 1..20 /product=“OTHER” /note= “Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACID OR CAN BEABSENT” Modified-site 21 /product= “OTHER” /note= “CYS AT POSITION 21CAN BE ABSENT” Modified-site 22 /product= “OTHER” /note= “Xaa ATPOSITION 22 CAN BE TYR OR TRP OR CAN BE ABSENT” Modified-site 23..42/product= “OTHER” /note= “Xaa AT POSITION 23-42 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” Modified-site 50..69 /product= “OTHER” /note= “Xaa ATPOSITIONS 50-69 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site75 /product= “OTHER” /note= “CYS AT POSITION 75 CAN BE ABSENT”Modified-site 76..95 /product= “OTHER” /note= “Xaa AT POSITIONS 76-95CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” 63 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Pro Lys Lys Lys Arg Lys 35 40 45 Val Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa ArgGly Asp Ser Pro Cys Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 117 amino acids aminoacid not relevant linear peptide Modified-site 1..20 /product= “OTHER”/note= “Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACID OR CAN BE ABSENT”Modified-site 21 /product= “OTHER” /note= “CYS AT POSITION 21 CAN BEABSENT” Modified-site 28 /product= “OTHER” /note= “CYS AT POSITION 28CAN BE ABSENT” Modified-site 29..48 /product= “OTHER” /note= “Xaa ATPOSITIONS 29-48 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site49 /product= “OTHER” /note= “CYS AT POSITION 49 CAN BE ABSENT”Modified-site 50..69 /product= “OTHER” /note= “Xaa AT POSITIONS 50-69CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 70 /product=“OTHER” /note= “Xaa AT POSITION 70 CAN BE TYR OR TRP OR CAN BE ABSENT”Modified-site 71..90 /product= “OTHER” /note= “Xaa AT POSITIONS 71-90CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 98..117 /product=“OTHER” /note= “Xaa AT POSITIONS 98-117 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” 64 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys Lys Phe Thr Ile Val Phe Cys Xaa XaaXaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 35 40 45 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Lys LysLys Arg Lys 85 90 95 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa 115 97 amino acids aminoacid not relevant linear peptide Modified-site 1..20 /product= “OTHER”/note= “Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 21 /product= “OTHER” /note= “CYS AT POSITION 21 CAN BEABSENT” Modified-site 28 /product= “OTHER” /note= “CYS AT POSITION 28CAN BE ABSENT” Modified-site 29..48 /product= “OTHER” /note= “Xaa ATPOSITIONS 29-48 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site49 /product= “OTHER” /note= “CYS AT POSITION 49 CAN BE ABSENT”Modified-site 50..69 /product= “OTHER” /note= “Xaa AT POSITIONS 50-69CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 70 /product=“OTHER” /note= “Xaa AT POSITION 70 CAN BE TYR OR TRP OR CAN BE ABSENT”Modified-site 78..97 /product= “OTHER” /note= “Xaa AT POSITIONS 78-97CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” 65 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys LysPhe Thr Ile Val Phe Cys Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Cys Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa XaaPro Lys Lys Lys Arg Lys Val Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa 99 amino acidsamino acid not relevant linear peptide Modified-site 1..20 /product=“OTHER” /note= “Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 21 /product= “OTHER” /note= “CYS AT POSITION 21CAN BE ABSENT” Modified-site 22 /product= “OTHER” /note= “Xaa ATPOSITION 22 CAN BE TYR, TRP OR CAN BE ABSENT” Modified-site 23..42/product= “OTHER” /note= “Xaa AT POSITIONS 23-42 CAN BE ANY AMINO ACIDSOR CAN BE ABSENT” Modified-site 51..70 /product= “OTHER” /note= “Xaa ATPOSITIONS 51-70 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site79 /product= “OTHER” /note= “CYS AT POSITION 79 CAN BE ABSENT”Modified-site 80..99 /product= “OTHER” /note= “Xaa FROM 80-99 CAN BE ANYAMINO ACIDS OR CAN BE ABSENT” 66 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Gly Pro Lys Lys Lys Arg 35 40 45 Lys Val Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Glu Ile LeuAsp Val Ser Pro Thr Cys Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa 78 amino acidsamino acid not relevant linear peptide Modified-site 1..20 /product=“OTHER” /note= “Xaa FROM 1-20 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 21 /product= “OTHER” /note= “CYS AT POSITION 21 CAN BEABSENT” Modified-site 22 /product= “OTHER” /note= “Xaa AT POSITION 22CAN BE TYR, TRP OR CAN BE ABSENT” Modified-site 30..49 /product= “OTHER”/note= “Xaa AT POSITIONS 30-49 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 58 /product= “OTHER” /note= “CYS AT POSITION 58 CAN BEABSENT” Modified-site 59..78 /product= “OTHER” /note= “Xaa AT POSITIONS59-78 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” 67 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa XaaCys Xaa Pro Lys Lys Lys Arg Lys Val Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Glu Ile LeuAsp Val Ser Pro Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 77 amino acids aminoacid not relevant linear peptide Modified-site 1..20 /product= “OTHER”/note= “Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACID OR CAN BE ABSENT”Modified-site 21 /product= “OTHER” /note= “CYS AT POSITION 21 CAN BEABSENT” Modified-site 28 /product= “OTHER” /note= “CYS AT POSITION 28CAN BE ABSENT” Modified-site 29..48 /product= “OTHER” /note= “Xaa ATPOSITION 29-48 CAN BE ANY AMINO ACID OR CAN BE ABSENT” Modified-site 57/product= “OTHER” /note= “CYS AT POSITION 57 CAN BE ABSENT”Modified-site 58..77 /product= “OTHER” /note= “Xaa AT POSITIONS 58-77CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” 68 Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys LysPhe Thr Ile Val Phe Cys Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Glu Ile Leu Asp Val ProSer Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 93 amino acids amino acid notrelevant linear peptide Modified-site 1..20 /product= “OTHER” /note=“Xaa AT POSITIONS 1-20 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 21 /product= “OTHER” /note= “Xaa AT POSITION 21 CAN BETYR, TRP OR CAN BE ABSENT” Modified-site 22..41 /product= “OTHER” /note=“Xaa AT POSITIONS 22-41 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”Modified-site 49..68 /product= “OTHER” /note= “Xaa AT POSITIONS 49-68CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 74..93 /product=“OTHER” /note= “Xaa AT POSITIONS 74-93 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” 69 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Lys Lys Lys ArgLys Val 35 40 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Arg Gly Asp Met Phe Xaa Xaa Xaa Xaa XaaXaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa85 90 94 amino acids amino acid not relevant linear peptideModified-site 1..20 /product= “OTHER” /note= “Xaa AT POSITIONS 1-20 CANBE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 21 /product= “OTHER”/note= “CYS AT POSITION 21 CAN BE ABSENT” Modified-site 22..41 /product=“OTHER” /note= “Xaa AT POSITIONS 22-41 CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 42 /product= “OTHER” /note= “Xaa AT POSITION 42CAN BE ANY AMINO ACID OR CAN BE ABSENT” Modified-site 50..69 /product=“OTHER” /note= “Xaa AT POSITIONS 50-69 CAN BE ANY AMINO ACID OR CAN BEABSENT” Modified-site 75..94 /product= “OTHER” /note= “Xaa AT POSITIONS75-94 CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” 70 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa XaaCys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Pro Lys Lys Lys Arg Lys 35 40 45 Val Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa XaaXaa Arg Gly Asp Met Phe Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 94 amino acids aminoacid not relevant linear peptide Modified-site 1..20 /product= “OTHER”/note= “Xaa CAN BE ANY AMINO ACID OR CAN BE ABSENT” Modified-site 21/product= “OTHER” /note= “Xaa CAN BE TYR, TRP OR CAN BE ABSENT”Modified-site 22..41 /product= “OTHER” /note= “Xaa CAN BE ANY AMINOACIDS OR CAN BE ABSENT” Modified-site 49..68 /product= “OTHER” /note=“Xaa CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 75..94/product= “OTHER” /note= “Xaa CAN BE ANY AMINO ACIDS OR CAN BE ABSENT”71 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 510 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 2025 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Lys Lys Lys Arg Lys Val 3540 45 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 5055 60 Xaa Xaa Xaa Xaa Gly Arg Gly Asp Ser Pro Xaa Xaa Xaa Xaa Xaa Xaa 6570 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 9094 amino acids amino acid not relevant linear peptide Modified-site1..20 /product= “OTHER” /note= “Xaa CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 21 /product= “OTHER” /note= “CYS CAN BE ABSENT”Modified-site 22 /product= “OTHER” /note= “Xaa CAN BE TYR, TRP OR CAN BEABSENT” Modified-site 23..42 /product= “OTHER” /note= “Xaa CAN BE ANYAMINO ACIDS OR CAN BE ABSENT” Modified-site 50..69 /product= “OTHER”/note= “Xaa CAN BE ANY AMINO ACIDS OR CAN BE ABSENT” Modified-site75..94 /product= “OTHER” /note= “Xaa CAN BE ANY AMINO ACIDS OR CAN BEABSENT” 72 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Lys Lys LysArg Lys 35 40 45 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Arg Gly Asp Ser Pro Xaa Xaa Xaa XaaXaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 85 90 116 amino acids amino acid not relevant linear peptideModified-site 1..20 /product= “other” /note= “Xaa CAN BE ANY AMINO ACIDOR CAN BE ABSENT” Modified-site 27 /product= “OTHER” /note= “CYS CAN BEABSENT” Modified-site 28..47 /product= “OTHER” /note= “Xaa CAN BE ANYAMINO ACID OR CAN BE ABSENT” Modified-site 48 /product= “OTHER” /note=“CYS CAN BE ABSENT” Modified-site 49..68 /product= “OTHER” /note= “XaaCAN BE ANY AMINO ACID OR CAN BE ABSENT” Modified-site 69 /product=“OTHER” /note= “Xaa AT POSITION 69 CAN BE TYR, TRP, OR CAN BE ABSENT”Modified-site 70..89 /product= “OTHER” /note= “Xaa CAN BE ANY AMINOACIDS OR CAN BE ABSENT” Modified-site 97..116 /product= “OTHER” /note=“Xaa CAN BE ANY AMINO ACID OR CAN BE ABSENT” 73 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa LysPhe Thr Ile Val Phe Cys Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 35 40 45 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Pro Lys Lys Lys Arg Lys Val 85 90 95 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa XaaXaa 115 97 amino acids amino acid not relevant linear peptideModified-site 1..20 /product= “OTHER” /note= “Xaa CAN BE ANY AMINO ACIDOR CAN BE ABSENT” Modified-site 21 /product= “OTHER” /note= “CYS CAN BEABSENT” Modified-site 28 /product= “OTHER” /note= “CYS CAN BE ABSENT”Modified-site 29..48 /product= “OTHER” /note= “Xaa CAN BE ANY AMINO ACIDOR CAN BE ABSENT” Active-site 49 /product= “OTHER” /note= “CYS CAN BEABSENT” Modified-site 50..69 /product= “OTHER” /note= “Xaa CAN BE ANYAMINO ACID OR CAN BE ABSENT” Modified-site 70 /product= “OTHER” /note=“Xaa CAN BE TYR, TRP OR CAN BE ABSENT” Modified-site 78..97 /product=“OTHER” /note= “Xaa CAN BE ANY AMINO ACID OR CAN BE ABSENT” 74 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 XaaXaa Xaa Xaa Cys Lys Phe Thr Ile Val Phe Cys Xaa Xaa Xaa Xaa 20 25 30 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 CysXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 XaaXaa Xaa Xaa Xaa Xaa Pro Lys Lys Lys Arg Lys Val Xaa Xaa Xaa 65 70 75 80Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 85 90 95Xaa 97 amino acids amino acid not relevant linear peptide Modified-site1..20 /product= “OTHER” /note= “Xaa CAN BE ANY AMINO ACID OR CAN BEABSENT” Modified-site 21 /product= “OTHER” /note= “Xaa CAN BE TYR, TRPOR CAN BE ABSENT” Modified-site 22..41 /product= “other” /note= “Xaa CANBE ANY AMINO ACID OR CAN BE ABSENT” Modified-site 50..69 /product=“OTHER” /note= “Xaa CAN BE ANY AMINO ACID OR CAN BE ABSENT”Modified-site 78..97 /product= “OTHER” /note= “Xaa CAN BE ANY AMINO ACIDOR CAN BE ABSENT” 75 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Pro LysLys Lys Arg Lys 35 40 45 Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Glu Ile Leu Asp Val Ser ProThr Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa 85 90 95 Xaa 77 amino acids amino acid not relevantlinear peptide Modified-site 1..20 /product= “OTHER” /note= “Xaa CAN BEANY AMINO ACIDS OR CAN BE ABSENT” Modified-site 21 /product= “OTHER”/note= “CYS CAN BE ABSENT” Modified-site 22 /product= “OTHER” /note=“Xaa CAN BE TYR, TRP OR CAN BE ABSENT” Modified-site 30..49 /product=“OTHER” /note= “Xaa CAN BE ANY AMINO ACID OR CAN BE ABSENT”Modified-site 58..77 /product= “OTHER” /note= “Xaa CAN BE ANY AMINO ACIDOR CAN BE ABSENT” 76 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys Xaa Pro Lys Lys Lys Arg LysVal Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa 35 40 45 Xaa Glu Ile Leu Asp Val Ser Pro Thr Xaa Xaa XaaXaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa 65 70 75 75 amino acids amino acid not relevant linear peptideModified-site 1..20 /product= “OTHER” /note= “Xaa CAN BE ANY AMINO ACIDOR CAN BE ABSENT” Modified-site 27 /product= “OTHER” /note= “CYS CAN BEABSENT” Modified-site 28..47 /product= “OTHER” /note= “Xaa CAN BE ANYAMINO ACID AND CAN BE ABSENT” Modified-site 56..75 /product= “OTHER”/note= “Xaa CAN BE ANY AMINO ACID OR CAN BE ABSENT” 77 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa XaaXaa Lys Phe Thr Ile Val Phe Cys Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu 35 40 45 Ile Leu AspVal Pro Ser Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 70 amino acids amino acid notrelevant circular peptide Modified-site 1..20 /product= “OTHER” /note=“Xaa CAN BE ANY CATIONIC AMINO ACIDS OR CAN BE ABSENT” Modified-site21..40 /product= “OTHER” /note= “Xaa CAN BE ANY AMINO ACIDS OR CAN BEABSENT” Modified-site 51..70 /product= “OTHER” /note= “Xaa CAN BE ANYAMINO ACIDS OR CAN BE ABSENT” 78 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa CysGlu Ile Leu Asp Val Pro Ser 35 40 45 Thr Cys Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa Xaa Xaa Xaa Xaa 65 70 71amino acids amino acid not relevant circular peptide Modified-site 1..20/product= “OTHER” /note= “Xaa CAN BE ANY CATIONIC AMINO ACID OR CAN BEABSENT” Modified-site 21..40 /product= “OTHER” /note= “Xaa CAN BE ANYAMINO ACID OR CAN BE ABSENT” Modified-site 52..71 /product= “OTHER”/note= “Xaa CAN BE ANY AMINO ACID OR CAN BE ABSENT” 79 Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Cys His Ala Ile Arg Gly Asp Thr 35 40 45 Phe Ala CysXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50 55 60 Xaa Xaa XaaXaa Xaa Xaa Xaa 65 70 14 amino acids amino acid not relevant linearpeptide NO NO Modified-site 14 /product= “OTHER” /note= “G AT POSITION14 CAN BE ABSENT” 80 Gly Lys Phe Thr Ile Val Phe Asp Asp Asp Asp Asp AspGly 1 5 10 28 amino acids amino acid not relevant linear peptide NO NO81 Lys Phe Thr Ile Val Phe Gly Gly Gly Leu Phe Glu Ala Ile Ala Glu 1 510 15 Phe Ile Glu Gly Gly Trp Glu Gly Leu Ile Glu Gly 20 25 27 aminoacids amino acid not relevant linear peptide NO NO 82 Gly Leu Phe GluAla Ile Ala Glu Phe Ile Glu Gly Gly Trp Glu Gly 1 5 10 15 Leu Ile GluGly Cys Lys Phe Thr Ile Val Phe 20 25 38 amino acids amino acid notrelevant linear peptide NO NO 83 Cys Gly Tyr Gly Gly Gly Gly Gly Gly ProLys Lys Lys Arg Lys Val 1 5 10 15 Gly Gly Gly Leu Phe Glu Ala Ile AlaGlu Phe Ile Glu Gly Gly Trp 20 25 30 Glu Gly Leu Ile Glu Gly 35 38 aminoacids amino acid not relevant linear peptide NO NO 84 Gly Leu Phe GluAla Ile Ala Glu Phe Ile Glu Gly Gly Trp Glu Gly 1 5 10 15 Leu Ile GluGly Gly Gly Tyr Gly Gly Gly Gly Gly Gly Pro Lys Lys 20 25 30 Lys Arg LysVal Gly Gly 35 20 amino acids amino acid not relevant linear peptide NONO 85 Lys Phe Thr Thr Ile Val Phe Cys Gly Tyr Gly Pro Lys Lys Lys Arg 15 10 15 Lys Val Gly Gly 20 19 amino acids amino acid not relevant linearpeptide NO NO 86 Cys Gly Tyr Gly Pro Lys Lys Lys Arg Lys Val Gly Gly LysPhe Thr 1 5 10 15 Ile Val Phe 30 amino acids amino acid not relevantlinear peptide NO NO Modified-site 30 /product= “OTHER” /note= “G ATPOSITION 30 CAN BE ABSENT” 87 Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys 1 5 10 15 Gly Lys Phe Thr Ile Val Phe Asp AspAsp Asp Asp Asp Gly 20 25 30 44 amino acids amino acid not relevantlinear peptide NO NO 88 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys 1 5 10 15 Lys Phe Thr Ile Val Phe Gly Gly Gly Leu PheGlu Ala Ile Ala Glu 20 25 30 Phe Ile Glu Gly Gly Trp Glu Gly Leu Ile GluGly 35 40 43 amino acids amino acid not relevant linear peptide NO NO 89Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 1015 Gly Leu Phe Glu Ala Ile Ala Glu Phe Ile Glu Gly Gly Trp Glu Gly 20 2530 Leu Ile Glu Gly Cys Lys Phe Thr Ile Val Phe 35 40 54 amino acidsamino acid not relevant linear peptide NO NO 90 Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Gly Leu Phe Glu AlaIle Ala Glu Phe Ile Glu Gly Gly Trp Glu Gly 20 25 30 Leu Ile Glu Gly GlyGly Tyr Gly Gly Gly Gly Gly Gly Pro Lys Lys 35 40 45 Lys Arg Lys Val GlyGly 50 36 amino acids amino acid not relevant linear peptide NO NO 91Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 1015 Lys Phe Thr Thr Ile Val Phe Cys Gly Tyr Gly Pro Lys Lys Lys Arg 20 2530 Lys Val Gly Gly 35 37 amino acids amino acid not relevant linearpeptide NO NO 92 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys 1 5 10 15 Gly Gly Cys Gly Tyr Gly Pro Lys Lys Lys Arg Lys ValGly Gly Lys 20 25 30 Phe Thr Ile Val Phe 35 56 amino acids amino acidnot relevant linear peptide NO NO 93 Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys 1 5 10 15 Gly Gly Cys Gly Tyr Gly Gly GlyGly Gly Gly Pro Lys Lys Lys Arg 20 25 30 Lys Val Gly Gly Gly Leu Phe GluAla Ile Ala Glu Phe Ile Glu Gly 35 40 45 Gly Trp Glu Gly Leu Ile Glu Gly50 55 24 amino acids amino acid not relevant linear peptide NO NO 94 SerSer Asp Asp Glu Ala Thr Ala Asp Ser Gln His Ser Thr Pro Pro 1 5 10 15Lys Lys Lys Arg Lys Val Gly Gly 20 40 amino acids amino acid notrelevant linear peptide NO NO 95 Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys 1 5 10 15 Ser Ser Asp Asp Glu Ala Thr Ala AspSer Gln His Ser Thr Pro Pro 20 25 30 Lys Lys Lys Arg Lys Val Gly Gly 3540 25 amino acids amino acid not relevant linear peptide NO NO 96 MetSer Tyr Tyr His His His His His His Asp Tyr Asp Ile Pro Thr 1 5 10 15Thr Glu Asn Leu Tyr Phe Gln Gly Ser 20 25 37 amino acids amino acid notrelevant linear peptide NO NO 97 Met Ser Tyr Tyr His His His His His HisAsp Tyr Asp Ile Pro Thr 1 5 10 15 Thr Glu Asn Leu Tyr Phe Gln Gly SerGly Tyr Gly Pro Lys Lys Lys 20 25 30 Arg Lys Val Gly Gly 35 30 aminoacids amino acid not relevant linear peptide NO NO 98 Met Ser Tyr TyrHis His His His His His Asp Tyr Asp Ile Pro Thr 1 5 10 15 Thr Glu AsnLeu Tyr Phe Gln Ser Lys Phe Thr Ile Val Phe 20 25 30 44 amino acidsamino acid not relevant linear peptide NO NO 99 Met Ser Tyr Tyr His HisHis His His His Asp Tyr Asp Ile Pro Thr 1 5 10 15 Thr Glu Asn Leu TyrPhe Gln Ser Gly Leu Phe Glu Ala Ile Ala Glu 20 25 30 Phe Ile Glu Gly GlyTrp Glu Gly Leu Ile Glu Gly 35 40 30 amino acids amino acid not relevantlinear peptide NO NO 100 Met Ser Tyr Tyr His His His His His His Asp TyrAsp Ile Pro Thr 1 5 10 15 Thr Glu Asn Leu Tyr Phe Gln Ser Arg Gly AspSer Pro Cys 20 25 30 10 amino acids amino acid not relevant linearpeptide NO NO 101 Met Ser Tyr Tyr His His His His His His 1 5 10 22amino acids amino acid not relevant linear peptide NO NO 102 Met Ser TyrTyr His His His His His His Gly Tyr Gly Pro Lys Lys 1 5 10 15 Lys ArgLys Val Gly Gly 20 16 amino acids amino acid not relevant linear peptideNO NO 103 Met Ser Tyr Tyr His His His His His His Lys Phe Thr Ile ValPhe 1 5 10 15 30 amino acids amino acid not relevant linear peptide NONO 104 Met Ser Tyr Tyr His His His His His His Gly Leu Phe Glu Ala Ile 15 10 15 Ala Glu Phe Ile Glu Gly Gly Trp Glu Gly Leu Ile Glu Gly 20 25 3016 amino acids amino acid not relevant linear peptide NO NO 105 Met SerTyr Tyr His His His His His His Arg Gly Asp Ser Pro Cys 1 5 10 15 6amino acids amino acid not relevant linear peptide NO NO 106 His His HisHis His His 1 5 16 amino acids amino acid not relevant linear peptide NONO 107 Gly Gly Gly Gly Gly Tyr Gly Pro Lys Lys Lys Arg Lys Val Gly Gly 15 10 15 12 amino acids amino acid not relevant linear peptide NO NO 108Gly Gly Tyr Gly Pro Lys Lys Lys Arg Lys Val Gly 1 5 10 14 amino acidsamino acid not relevant linear peptide NO NO 109 Gly Cys Gly Tyr Gly ProLys Lys Lys Arg Lys Val Gly Gly 1 5 10 9 amino acids amino acid notrelevant linear peptide NO NO 110 Gly Gly Arg Gly Asp Met Phe Gly Gly 15 22 amino acids amino acid not relevant linear peptide NO NO 111 GlyTyr Gly Pro Lys Lys Lys Arg Lys Val Gly Gly Gly Gly Gly Arg 1 5 10 15Gly Asp Met Phe Gly Gly 20 20 amino acids amino acid not relevant linearpeptide NO NO 112 Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn GlyTrp Glu Gly 1 5 10 15 Met Ile Asp Gly 20 20 amino acids amino acid notrelevant linear peptide NO NO 113 Gly Leu Phe Gly Ala Ile Ala Gly PheIle Glu Asn Gly Trp Glu Gly 1 5 10 15 Leu Ile Glu Gly 20 20 amino acidsamino acid not relevant linear peptide NO NO 114 Gly Leu Phe Lys Ala IleAla Lys Phe Ile Lys Gly Gly Trp Lys Gly 1 5 10 15 Leu Ile Lys Gly 20 12amino acids amino acid <Unknown> not relevant peptide NO NO 115 Gly TyrGly Pro Lys Lys Lys Arg Lys Val Gly Gly 1 5 10 31 amino acids amino acidnot relevant linear peptide NO NO 116 Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Gly Gly Cys Gly Tyr Gly ProLys Lys Lys Arg Lys Val Gly Gly 20 25 30 36 amino acids amino acid notrelevant linear peptide NO NO 117 Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys 1 5 10 15 Cys Gly Tyr Gly Pro Lys Lys LysArg Lys Val Gly Gly Gly Gly Arg 20 25 30 Gly Asp Ser Pro 35 25 aminoacids amino acid not relevant linear peptide NO NO 118 Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Gly Gly ArgGly Asp Ser Pro Cys Gly 20 25 25 amino acids amino acid not relevantlinear peptide NO NO 119 Gly Arg Gly Asp Ser Pro Cys Gly Gly Lys Lys LysLys Lys Lys Lys 1 5 10 15 Lys Lys Lys Lys Lys Lys Lys Lys Lys 20 25 22amino acids amino acid not relevant linear peptide NO NO 120 Cys Gly TyrGly Pro Lys Lys Lys Arg Lys Val Gly Gly Gly Gly Arg 1 5 10 15 Gly AspSer Pro Cys Gly 20

We claim:
 1. A composition for transfecting a cell which comprises oneor more nucleic acid molecules, one or more peptides or proteins, andone or more transfection agents.
 2. The composition of claim 1, whereinsaid composition comprises two or more peptides and/or proteins.
 3. Thecomposition of claim 1, wherein said composition comprises two or moretransfection agents.
 4. The composition of claim 1, wherein saidcomposition comprises a peptide- or protein-nucleic acid complex.
 5. Thecomposition of claim 4, wherein said peptide- or protein-nucleic acidcomplex comprises two or more peptides, or proteins or both.
 6. Thecomposition of claim 1, wherein said transfection agent comprises one ormore cationic lipids.
 7. The composition of claim 6, wherein saidtransfection agent further comprises one or more neutral lipids.
 8. Thecomposition of claim 1, wherein said transfection agent comprises one ormore dendrimers.
 9. The composition of claim 8, wherein saidtransfection agent further comprises one or more lipids.
 10. Thecomposition of claim 6, wherein said cationic lipids comprise one ormore monovalent cationic lipids.
 11. The composition of claim 10,wherein said monovalent cationic lipids are selected from the groupconsisting of DOTMA, DOTAP, DMRIE, and DDAB.
 12. The composition ofclaim 6, wherein said cationic lipids comprise one or more polyvalentcationic lipids.
 13. The composition of claim 12, wherein saidpolyvalent cationic lipids are selected from the group consisting ofDOSPA, DOSPER, DOGS, TMTPS, TMTOS, TMTLS, TMTMS, and TMDOS.
 14. Thecomposition of claim 7, wherein said neutral lipids are selected fromthe group consisting of DOPE, DPhPE, and cholesterol.
 15. Thecomposition of claim 8, wherein said dendrimers are selected from thegroup consisting of dense star dendrimers, PAMAM dendrimers, NH₃ coredendrimers, ethylenediamine core dendrimers, dendrimers of generation 5or higher, dendrimers with substituted groups, dendrimers comprising oneor more amino acids, grafted dendrimers and activated dendrimers. 16.The composition of claim 1, wherein one or more of said transfectionagents are convalently linked to one or more of said peptides and/orproteins.
 17. The composition of claim 6, wherein one or more of saidcationic lipids are covalently linked to one or more of said peptidesand/or proteins.
 18. The composition of claim 7, wherein one or more ofsaid neutral lipids are covalently linked to one or more of saidpeptides and/or proteins.
 19. The composition of claim 8, wherein one ormore of said dendrimers are covalently linked to one or more of saidpeptides and/or proteins.
 20. The composition of claim 1, wherein saidpeptides and/or proteins are derived from animal, bacterial, viralpeptides and/or proteins.
 21. The composition of claim 1, wherein saidpeptides and/or proteins are conjugated to one or more nucleic acidbinding groups.
 22. The composition of claim 21, wherein said nucleicacid binding groups comprise at least one polyamine.
 23. The compositionof claim 22, wherein said nucleic acid binding group comprises at leastone spermine.
 24. The composition of claim 1, wherein at least one ofsaid peptide and/or protein is a nuclear localization protein orpeptide.
 25. The composition of claim 1, wherein at least one of saidpeptide and/or protein is a fusagenic peptide or protein.
 26. Thecomposition of claim 1, wherein at least one of said peptide and/orprotein is a receptor-ligand peptide or protein.
 27. The composition ofclaim 1, wherein at least one of said peptide and/or protein is atransport peptide or protein.
 28. The composition of claim 20, whereinat least one of said peptide and/or protein is a viral peptide orprotein.
 29. The composition of claim 28, wherein said virus is selectedfrom the group consisting of an influenza virus, a vesicular stomatitisvirus, an adenovirus, an alphavirus, a Semliki Forest Virus, a hepatitisvirus, a herpes virus, an HIV virus, and a simian virus.
 30. Thecomposition of claim 1, further comprising DEAE-dextran, chloroquine orcombinations thereof.
 31. The composition of claim 1, wherein at leastone of said peptide and/or protein is selected from the group consistingof an insulin, a transferrin, a epidermal growth factor, a fibroblastgrowth factor, a lactoferrin, a fibronectin, an adenovirus penton base,Knob, and hexon protein, a vesicular stomatitis virus glycoprotein, aSemliki Forest Virus core protein, a influenza hemagglutinin, ahepatitis B core protein, an HIV Tat protein, a herpes simplex virusVP22 protein, a histone protein, a high mobility group protein, andinvasin protein, and internalin protein, an endotoxin, a diptheriatoxin, a shigella toxin, a melittin, a magainin, a gramicidin, acecrophin, a defensins, a protegrins, a tachyplesins, a thionins, aindolicidin, a bactenecin, a drosomycin, a apidaecins, a cathelicidin, abacteriacidal-permability-increasing protein, a nisin, and a buforin,and fragments thereof.
 32. The composition of claim 1, wherein saidcomposition is capable of transfecting a primary cell culture, apassaged cell culture or a cell line.
 33. The composition of claim 32,wherein said cell line is a human cell line.
 34. The composition ofclaim 32, wherein said cell line is an animal cell line.
 35. Thecomposition of claim 32, wherein said cell line is a fibroblast.
 36. Thecomposition of claim 1, wherein at least one of said peptides and/orproteins comprise multimers of the same or different peptides orproteins.
 37. The composition of claim 1, wherein said peptide and/orprotein comprises one or more amino acid derivatives or analogues. 38.The composition of claim 1, wherein at least one of said peptides and/orproteins comprises two or more functions selected from the groupconsisting of fusagenic, nuclear localization, transport,receptor-ligand and cell adhesion.
 39. A pharmaceutical compositioncomprising an amount of the composition of claim 1 effective fortransfection of a targeted cell or tissue and a pharmaceutical carrier.40. A therapeutic composition comprising an amount of the composition ofclaim 1 effective for transfection of a targeted cell or tissue with aselected therapeutic nucleic acid.
 41. A diagnostic compositioncomprising an amount of the composition of claim 1 effective fortransfection of a targeted cell or tissue with a selected diagnosticnucleic acid.
 42. A composition for transfecting a cell which comprisesa component of transfection agent covalently linked to a peptide orprotein.
 43. The composition of claim 42 wherein the component of atransfection agent is a lipid.
 44. The composition of claim 42 whereinthe component of a transfection agent is a cationic lipid.
 45. Thecomposition of claim 42 wherein the component of a transfection agent isa neutral lipid.
 46. The composition of claim 42 wherein the componentof a transfection agent is a dendrimer.
 47. The composition of claim 42further comprising a receptor-ligand protein.
 48. A composition fortransfecting a cell obtained by combining one or more nucleic acidmolecules, one or more peptides or proteins, and one or moretransfection agents.
 49. A composition for transfecting a cell of claim48 obtained by first forming a peptide- or protein-nucleic acid complexfollowed by addition of a transfection agent capable of aggregating thepeptide-or protein-nucleic acid complex.
 50. The composition of claim 49wherein after the peptide-or protein- nucleic acid complex is formed,said complex is added to a mixture of a cationic lipid and a neutrallipid.
 51. A method for transfecting a cell with a nucleic acid, themethod comprising the step of contacting the cell with the transfectioncomposition of claim
 1. 52. A method for transfecting a cell with anucleic acid, the method comprising the step of contacting the cell withthe transfection composition of claim
 17. 53. A method for transfectinga cell with a nucleic acid, the method comprising the step of contactingthe cell with the transfection composition of claim
 31. 54. A method fortransfecting a cell with a nucleic acid, the method comprising the stepof contacting the cell with the transfection composition of claim 48.55. A method for transfecting a cell with a nucleic acid, the methodcomprising the steps: (a) admixing one or more peptides or proteins witha nucleic acid to form a peptide-nucleic acid complex or aprotein-nucleic acid complex; (b) adding a transfection agent to thecomplex from step (a) to obtain an aggregate of the transfection agentand said complex; and (c) contacting said cell with the aggregate fromstep (b).
 56. The method of claim 55 wherein the peptides or proteinscomprises a sub-cellular localization signal sequence, a nuclearlocalization signal sequence, a fusagenic sequence, a transport ortrafficking sequence, receptor-ligand sequence or a cell adhesionsequence.
 57. The method of claim 56 wherein the peptide or protein ismodified by covalent bonding to a nucleic acid-binding group.
 58. Themethod of claim 57 wherein the nucleic acid-binding group is a spermine.59. The method of claim 58 wherein the peptide is Sp-NLS, Sp-NLSNLS,Sp-NLSRGD, Opf-GG-1, Opf-GG-2, Opf-GG-2-CYS or Sp-Tat.
 60. The method ofclaim 55 wherein the transfection agent comprises a dendrimer.
 61. Themethod of claim 60 wherein the transfection agent comprises an activateddendrimer.
 62. The method of claim 61 wherein the dendrimer is selectedfrom the group of GX(NH3) or GX(EDA) dendrimers where X is an integerfrom about 5 to about
 10. 63. The method of claim 60 wherein thedendrimer is conjugated to an arginine or a lysine.
 64. A transfectionreagent kit which comprises a transfection agent and a peptide orprotein or a modified peptide or protein capable of enhancingtransfection of the transfection agent.
 65. The kit of claim 64 whichcomprises a cationic lipid transfection agent.
 66. The kit of claim 65wherein the cationic lipid transfection agent is selected from the group“LIPOFECTAMINE”, “LIPOFECTIN”, “LIPOFECTACE”, “CELLFECTIN”,“MULTIFECTOR”, or “TRANSFECTIN”.
 67. The kit of claim 66 wherein thepeptide is Sp-NLSNLS.
 68. The kit of claim 66 wherein the peptide isSp-Tat.
 69. The kit of claim 64 which comprises a dendrimer transfectionagent.
 70. The kit of claim 69 wherein the dendrimer is a dense stardendrimer or an activated dendrimer.
 71. A kit of claim 64 that is adiagnostic kit and which further comprises a diagnostic nucleic acid.72. A peptide comprising an NLS sequence modified by covalent bonding toa nucleic acid-binding group.
 73. The modified peptide of claim 72 whichcomprises a dimer or multimer of an NLS sequence.
 74. The modifiedpeptide of claim 73 wherein the nucleic acid-binding group is aspermine.
 75. A peptide comprising a Tat sequence modified by covalentbonding to a nucleic acid-binding group.
 76. The modified peptide ofclaim 75 which comprises a dimer or multimer of a Tat sequence.
 77. Themodified peptide of claim 76 wherein the nucleic acid-binding group is aspermine.